Treatment of specific cardiovascular conditions with nitrite

ABSTRACT

It has been surprisingly discovered that administration of nitrite to subjects causes a reduction in blood pressure and an increase in blood flow to tissues. The effect is particularly beneficial, for example, to tissues in regions of low oxygen tension. This discovery provides useful treatments to regulate a subject&#39;s blood pressure and blood flow, for example, by the administration of nitrite salts. Provided herein are methods of administering a pharmaceutically-acceptable nitrite salt to a subject, for treating, preventing or ameliorating a condition selected from: (a) ischemia-reperfusion injury (e.g., hepatic or cardiac or brain ischemia-reperfusion injury); (b) pulmonary hypertension (e.g., neonatal pulmonary hypertension); or (c) cerebral artery vasospasm.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/563,683, filedOct. 4, 2006, which is a §371 U.S. national stage of PCT/US2004/022232,filed Jul. 9, 2004, which was published in English under PCT Article2(2), and which in turn claims the benefit of U.S. ProvisionalApplication No. 60/485,959, filed Jul. 9, 2003, and U.S. ProvisionalApplication No. 60/511,244, filed Oct. 14, 2003. All of the above-listedapplications are incorporated herein by reference in their entirety.

GOVERNMENT INTEREST STATEMENT

This invention was made with government support under Grant Nos. HL58091and HL70146, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

The last decade has seen an increase in the understanding of thecritical role nitric oxide as a blood vessel dilator contributing to theregulation of blood flow and cardiovascular homeostasis. Nitric oxidemay be oxidized in blood to nitrite (NO₂—), an anion considered to be aninert metabolic end product of such nitric oxide oxidation. In vivoplasma levels of nitrite have been reported to range from 150 to 1000nM, and the nitrite concentration in aortic ring tissue has beenreported to be in excess of 10,000 nM (Rodriguez et al., Proc Natl AcadSci USA, 100, 336-41, 2003; Gladwin et al., Proc Natl Acad Sci USA, 97,9943-8, 2000; and Rassaf et al., Nat Med, 9, 481-3, 2003). Thispotential storage pool for NO is in excess of plasma S-nitrosothiols,which have been reported to be less than 10 nM in human plasma (Rassafet al., Nat Med, 9, 481-3, 2003; Rassaf et al., Free Radic Biol Med, 33,1590-6, 2002; Rassaf et al., J Clin Invest, 109, 1241-8, 2002; andSchechter et al., J Clin Invest, 109, 1149-51, 2002). Mechanisms havebeen proposed for the in vivo conversion of nitrite to NO, for example,by enzymatic reduction by xanthine oxidoreductase or by non-enzymaticdisproportionation/acidic reduction (Millar et al., Biochem Soc Trans,25, 528S, 1997; Millar et al., FEBS Lett, 427, 225-8, 1998; Godber etal., J Biol Chem, 275, 7757-63, 2000; Zhang et al., Biochem Biophys ResCommun, 249, 767-72, 1998 [published erratum appears in Biochem BiophysRes Commun 251, 667, 1998]; Li et al., J Biol Chem, 276, 24482-9, 2001;Li et al., Biochemistry, 42, 1150-9, 2003; Zweier et al., Nat Med, 1,804-9, 1995; Zweier et al., Biochim Biophys Acta, 1411, 250-62, 1999;and Samouilov et al., Arch Biochem Biophys, 357:1-7, 1998).

Arterial-to-venous gradients of nitrite across the human forearm at restand during regional NO synthase inhibition have been observed, withincreased consumption of nitrite occurring with exercise (Gladwin etal., Proc Natl Acad Sci USA, 97, 9943-8, 2000; Gladwin et al., Proc NatlAcad Sci USA, 97, 11482-11487, 2000; and Cicinelli et al., Clin Physiol,19:440-2, 1999). Kelm and colleagues have reported that largeartery-to-vein gradients of nitrite form across the human forearm duringNO synthase inhibition (Lauer et al., Proc Natl Acad Sci USA, 98,12814-9, 2001). Unlike the more simple case of oxygen extraction acrossa vascular bed, nitrite may be both consumed, as evidenced byartery-to-vein gradients during NO synthase inhibition and exercise, andproduced in the vascular bed by endothelial nitric oxidesynthase-derived NO reactions with oxygen.

At high concentrations, nitrite has been reported to be a vasodilator invitro (Ignarro et al., Biochim Biophys Acta, 631, 221-31, 1980; Ignarroet al., J Pharmacol Exp Ther, 218, 739-49, 1981; Moulds et al., Br JClin Pharmacol, 11, 57-61, 1981; Gruetter et al., J Pharmacol Exp Ther,219, 181-6, 1981; Matsunaga et al., J Pharmacol Exp Ther, 248, 687-95,1989; and Laustiola et al., Pharmacol Toxicol, 68, 60-3, 1991). Thelevels of nitrite shown to vasodilate in vitro have always been inexcess of 100,000 nM (100 μM) and usually at millimolar concentrations.

Consistent with the high concentrations of nitrite required tovasodilate in vitro, when Lauer and colleagues infused nitrite into theforearm circulation of human subjects, they reported no vasodilatoryeffects, even with concentrations of 200 μM in the forearm (Lauer etal., Proc Natl Acad Sci USA, 98, 12814-9, 2001). Lauer et al. reportedthat a “complete lack of vasodilator activity of intraarterial infusionsof nitrite clearly rules out any role for this metabolite in NOdelivery” and concluded that “physiological levels of nitrite arevasodilator-inactive.” Furthermore, Rassaf and colleagues also failed tofind a vasodilatory effect in humans following infusion of nitrite(Rassaf et al., J Clin Invest, 109, 1241-8, 2002). Thus, in vivo studieshave concluded that physiological levels of nitrites do not serve as asource for NO, and that physiological levels of nitrites do not have arole in regulating blood pressure.

Historically, nitrite has been used as a treatment for cyanidepoisoning. High concentrations are infused into a subject sufferingcyanide poisoning in order to oxidize hemoglobin to methemoglobin, whichwill bind cyanide. These high concentrations of nitrite produceclinically significant methemoglobinemia, potentially decreasing oxygendelivery. While these high concentrations of nitrite have been shown todecrease blood pressure in humans, the amount of methemoglobin formedprecluded a use for nitrite in the treatment of other medicalconditions.

Therefore, the state of the art was that nitrite was not a significantvasodilator at concentrations below 100 μM in vitro, and even wheninfused into humans at concentrations of 200 μM in the forearm. It wasalso the state of the art that nitrite was not converted to nitric oxidein the human blood stream.

SUMMARY OF THE DISCLOSURE

It has been surprisingly discovered that administration ofpharmaceutically-acceptable salts of nitrite is useful in the regulationof the cardiovascular system. It has also been surprisingly discoveredthat nitrite is reduced to nitric oxide in vivo, and that the nitricoxide produced thereby is an effective vasodilator. These effectssurprisingly occur at doses that do not produce clinically significantmethemoglobinemia. These discoveries now enable methods to prevent andtreat conditions associated with the cardiovascular system, for example,high blood pressure, pulmonary hypertension, cerebral vasospasm andtissue ischemia-reperfusion injury. These discoveries also providemethods to increase blood flow to tissues, for example, to tissues inregions of low oxygen tension. It is particularly surprising that thenitrite does not need to be applied in an acidified condition in orderfor it to be effective in regulating the cardiovascular system, and moreparticularly to act as a vasodilator in vivo.

It has now been surprisingly discovered by the inventors that nitritecan serve as a vasodilator in humans at much lower concentrations (aslow as 0.9 μM) than have been used in the past for cyanide poisoning.The mechanism is believed to involve a reaction of nitrite withdeoxygenated hemoglobin and red blood cells, to produce the vasodilatinggas nitric oxide. This potent biological effect is observed at doses ofnitrite that do not produce clinically significant methemoglobinemia(for instance, less than 20%, more preferably less than 5% methemoglobinin the subject).

It has been discovered that nitrite is converted to nitric oxide invivo, and that the nitric oxide produced thereby is an effectivevasodilator. Further, it has been surprisingly discovered thatadministration of nitrite, for instance a pharmaceutically-acceptablesalt of nitrite, to a subject causes a reduction in blood pressure andan increase in blood flow to tissues, for example, to tissues in regionsof low oxygen tension. These discoveries now enable useful methods toregulate the cardiovascular system, for instance to prevent and treatmalconditions associated with the cardiovascular system, for example,high blood pressure, or organs, tissues, or systems suffering a lack ofor inadequate blood flow. Non-limiting examples of contemplatedmalconditions include stroke, heart disease, kidney disease and failure,eye damage including hypertensive retinopathy, diabetes, and migraines.

In one example embodiment, the present disclosure provides a method fordecreasing a subject's blood pressure or increasing blood flow,including in a particular embodiment administering to the subject sodiumnitrite at about 36 μmoles per minute into the forearm brachial artery.

The present disclosure additionally provides a method for increasingblood flow to a tissue of a subject, including administering to thesubject an effective amount of pharmaceutically-acceptable nitrite, suchas a salt thereof, so as to increase blood flow to a tissue of thesubject. The blood flow may be specifically increased in tissues inregions of low oxygen tension. The present disclosure also provides amethod for decreasing a subject's blood pressure, comprisingadministering to the subject an effective amount ofpharmaceutically-acceptable nitrite so as to decrease the subject'sblood pressure.

The present disclosure further provides a method for treating a subjecthaving a condition associated with elevated blood pressure, includingadministering to the subject an effective amount ofpharmaceutically-acceptable nitrite so as to treat at least one vascularcomplication associated with the elevated blood pressure.

Also provided is a method for treating a subject having a hemolyticcondition, including administering to the subject an effective amount ofpharmaceutically-acceptable nitrite so as to treat at least one vascularcomplication associated with the hemolytic condition.

The disclosure further provides a method for treating a subject having acondition associated with elevated blood pressure in the lungs, e.g.pulmonary hypertension, including administering to the subject aneffective amount of pharmaceutically-acceptable nitrite. In someembodiments, this includes treating a subject having neonatal pulmonaryhypertension. In some embodiments, this includes treating a subjecthaving primary and/or secondary pulmonary hypertension. In someembodiments for treating subjects having a condition associated withelevated blood pressure in the lungs, the nitrite is nebulized.

Also contemplated herein are methods for treating, ameliorating, orpreventing other conditions of or associated with blood flow, includingvasospasm, stroke, angina, revascularization of coronary arteries andother arteries (peripheral vascular disease), transplantation (e.g., ofkidney, heart, lung, or liver), treatment of low blood pressure (such asthat seen in shock or trauma, surgery and cardiopulmonary arrest) toprevent reperfusion injury to vital organs, cutaneous ulcers (e.g., withtopical, non-acidified nitrite salt), Raynauds phenomenon, treatment ofhemolytic conditions (such as sickle cell, malaria, TTP, and HUS),hemolysis caused by immune incompatibility before and after birth, andother conditions listed herein.

Also provided herein are methods of administering apharmaceutically-acceptable nitrite salt to a subject, for treating,preventing or ameliorating a condition selected from: (a)ischemia-reperfusion injury (e.g., hepatic or cardiac or brainischemia-reperfusion injury); (b) pulmonary hypertension (e.g., neonatalpulmonary hypertension); or (c) cerebral artery vasospasm. Alsocontemplated are methods for treatment, prevention, and/or ameliorationof gestational or fetal cardiovascular malconditions.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B are a pair of graphs, depicting hemodynamic and metabolicmeasurements at baseline and during exercise in 18 subjects. FIG. 1Ashows effects on each of the indicated values without inhibition of NOsynthesis. FIG. 1B shows effects with inhibition of NO synthesis. Key:MAP—mean arterial pressure, mmHg; FBF—forearm blood flow, mL/min/100 mL;O₂ saturation, %; pO₂—venous oxyhemoglobin saturation, partial pressureof oxygen, mmHg; pH, units; *=p<0.05 vs. Baseline 1 or 2, respectively;**=p<0.01 vs. Baseline 1 or 2, respectively; †=p<0.05 vs. Baseline 1;††=p<0.01 vs. Initial Exercise.

FIGS. 2A-2B are a pair of graphs, depicting effects of infusion ofsodium nitrite in bicarbonate-buffered normal saline into the brachialarteries of 18 healthy subjects. FIG. 2A shows effects on each of theindicated values without inhibition of NO synthesis. FIG. 2B showseffects with inhibition of NO synthesis. Key as for FIG. 1, plus:Nitrite—venous nitrite, μM; NO-heme—venous iron-nitrosyl-hemoglobin, μM;and MetHb—venous methemoglobin, %; +=p<0.01 vs. Initial Exercise.

FIGS. 3A-3D are a series of graphs, illustrating the effects of infusionof low-dose sodium nitrite into the brachial arteries of 10 healthysubjects at baseline and during exercise, without and with inhibition ofNO synthesis. FIG. 3A shows forearm blood flow at baseline and followinga five-minute infusion of NaNO₂. FIG. 3B shows forearm blood flow withand without low-dose nitrite infusion at baseline and during L-NMMAinfusion with and without exercise stress. FIG. 3C shows venous levelsof nitrite from the forearm circulation at the time of blood flowmeasurements. FIG. 3D shows venous levels of S-nitroso-hemoglobin (S-NO)and iron-nitrosyl-hemoglobin (Hb-NO) at baseline and following nitriteinfusion during exercise stress.

FIGS. 4A-4B are a pair of graphs, showing formation of NO-hemoglobinadducts. FIG. 4A shows formation of iron-nitrosyl-hemoglobin andS-nitroso-hemoglobin, comparing baseline, with nitrite infusion, andnitrite infusion with exercise. FIG. 4B compares formation ofNO-hemoglobin adducts with hemoglobin-oxygen saturation in the humancirculation, during nitrite infusion.

FIG. 5A shows NO release following nitrite injections into solutions ofPBS (“PBS”), deoxygenated red blood cells (“deoxy-RBC”), and oxygenatedred blood cells (“oxy-RBC”). FIG. 5B shows the rate of NO formation fromnitrite mixed with PBS (first bar in each set), and oxygenated anddeoxygenated red blood cells (second and third bar in each set,respectively).

FIGS. 6A-6F show nitrite therapy in hepatic ischemia-reperfusion injury.FIG. 6A illustrates the experimental protocol used for murine model ofhepatic ischemia-reperfusion injury. FIG. 6B is a graph showing serumAST levels in mice following hepatic ischemia-reperfusion. *p<0.05 vs.vehicle (0 μM) and **p<0.01 vs. vehicle (0 μM) FIG. 6C is a graphshowing serum ALT levels in mice following hepatic ischemia-reperfusion.*p<0.05 vs. vehicle (0 μM) and **p<0.01 vs. vehicle (0 μM) FIG. 6D is arepresentative photomicrographs of hepatic histopathology following 45minutes of ischemia and 24 hours of reperfusion. FIG. 6E is a bar graphshowing pathological scoring of hepatic tissue samples following 45minutes of ischemia and 24 hours of reperfusion. FIG. 6F is a bar graphshowing hepatocellular apoptosis as measured by TUNEL staining following45 minutes of ischemia and 24 hours of reperfusion. **p<0.001 vs. I/Ralone group

FIGS. 7A-7E show nitrite therapy in myocardial ischemia-reperfusioninjury. FIG. 7A illustrates the experimental protocol used formyocardial ischemia-reperfusion studies in mice. FIG. 7B is arepresentative photomicrographs of the murine hearts following 30minutes of myocardial ischemia and reperfusion. FIG. 7C is a bar graphcomparing myocardial area-at-risk (AAR) per left ventricle (LV), infarctsize (INF) per AAR, and infarct per left ventricle in mice treated withnitrate or nitrite. FIG. 7D is a bar graph comparing myocardial ejectionfraction at baseline and following 45 minutes of myocardial ischemia and48 hours of reperfusion. FIG. 7E is a bar graph comparing leftventricular fractional shortening at baseline and following 45 minutesof myocardial ischemia and 48 hours of reperfusion.

FIGS. 8A-8D are a series of graphs, illustrating blood and liver tissuelevels of nitrite, RSNO and RxNO. FIG. 8A shows blood nitrite, RSNO, andRxNO levels (μmol/L) in animals (n=3-5 per group) subjected to shamhepatic ischemia-reperfusion (I/R) or hepatic ischemia and either 1 or30 minutes of reperfusion. **p<0.001 vs. sham FIG. 8B shows liver tissuenitrite levels in mice (n=3-5 per group) subjected to hepaticischemia-reperfusion (I/R) injury. FIG. 8C shows liver tissue RSNOlevels (μmol/L) in mice (n=3-5 per group) subjected to hepatic ischemiaand varying periods of reperfusion. FIG. 8D shows hepatic tissue RxNOlevels (μmol/L) following hepatic ischemia and reperfusion in mice(n=3-5 per group).

FIGS. 9A-9D illustrate nitrite mediated hepatoprotection and the nitricoxide and heme oxygenase-1 signaling pathways. FIG. 9A is a graph,comparing serum aspartate aminotransferase (AST) levels in micereceiving saline vehicle, nitrite (24 μM), the nitric oxide (NO)scavenger PTIO, or nitrite (24 μM)+PTIO. **p<0.01 vs. the vehicle group.FIG. 9B is a graph comparing serum levels of AST in eNOS deficient (−/−)mice receiving saline vehicle or sodium nitrite (24 μM). FIG. 9C is animage showing hepatic protein levels of heme oxygenase-1 (HO-1)determined using western blot analysis in sham operated animals and inanimals subjected to hepatic ischemia (45 minutes) and reperfusion (5hours). FIG. 9D is a graph comparing serum AST levels in mice treatedwith nitrite (24 μM) or the HO-1 inhibitor zinc deuteroporphyrin bisglycol (ZnDPBG) in the setting of hepatic ischemia reperfusion injury.

FIGS. 10A-10C show the effects of nitrite anion inhalation in newbornhypoxic lambs (n=7) (FIG. 10A) on hemodynamic and metabolicmeasurements. After a hypoxic gas mixture (FiO₂=0.12) had been startedat time 0, nitrite by aerosol reduced pulmonary artery pressure (PAP)from hypoxic levels by 63+/−3% (P<0.01 versus hypoxic baseline) withlittle change in mean arterial pressure (MAP), cardiac output, ormethemoglobin levels, but a marked increase in exhaled NO (P<0.01compared to baseline). FIG. 10B illustrates the effect of salineinhalation on pulmonary artery pressure in hypoxic lambs (n=7). FIG. 10Cis a multipanel graph, showing maximal effects of nitrite nebulizationas compared to saline nebulization on PAP, MAP, and exhaled NO (eNO).Data are mean±SEM.

FIG. 11 illustrates effects of nitrite anion inhalation in newborn lambsduring stable, normoxic (SaO₂˜99%) pulmonary hypertension induced by theinfusion of an endoperoxide analog of thromboxane (U46619) (n=6). Afterinfusion of U46619 was started at time 0, nitrite by aerosol reducedpulmonary artery pressure (PAP) from infusion baseline level by 23±6%(P<0.05 compared to infusion baseline) with no measurable change in meanarterial pressure (MAP) and with a moderate increase in exhaled NO(P<0.01 compared to baseline).

FIG. 12A compares the change in pulmonary arterial pressure (PAP),exhaled NO, and iron-nitrosyl-hemoglobin as measured by bothchemiluminescence and electron paramagnetic resonance (EPR) afternitrite inhalation in animals with pulmonary hypertension induced witheither hypoxia or infusion of the thromboxane analog U46199. Data foriron-nitrosyl-hemoglobin, measured by areas of output peaks aftertri-iodide based reductive chemiluminescence (FIG. 12B) and by depth ofpeak at 3350 Gauss in electron paramagnetic resonance (EPR) (FIG. 12C;red line: drug induced, blue line: hypoxic) measured 20 minutes afternitrite inhalation was begun. FIG. 12D shows change in mean pulmonaryartery pressure during hypoxia after inhalation of nebulized sodiumnitrite was related to blood pH, with increased vasodilation associatedwith decreasing pH (r=0.57 P=0.055). Data are mean±SEM.

FIGS. 13A-13E show duration of effect of NO gas inhalation (n=7) (FIG.13A) or nitrite nebulization (n=7) (FIG. 13B) on hemodynamic andmetabolic measurements during hypoxic-induced pulmonary hypertension.Treatment with nitrite aerosol resulted in a rapid sustained reductionin hypoxic-induced pulmonary vasoconstriction and a graded increase inexhaled NO gas concentration with no change in mean arterial bloodpressure. These results are contrasted to the rapid return in pulmonaryartery pressure to hypoxic baseline after termination of inhaled NO gas(FIG. 13A). Methemoglobin (Met Hb) concentrations increased from2.1±0.1% during baseline to 2.8±0.2% after nitrite nebulization(P<0.05). Note that the exhaled nitric oxide concentrations in FIG. 13Areach the limit of detection during administration of inhaled nitricoxide (20 ppm). FIG. 13C shows the change in pulmonary artery pressure(PAP) after aerosolization of nebulized nitrite and during the remaininghour of hypoxia following the termination of nitrite nebulization. FIG.13D shows the arterial plasma nitrite concentrations during the courseof the experiment. FIG. 13E shows the relationship between pulmonaryartery pressure and exhaled NO after nitrite nebulization duringhypoxia. Data are mean±SEM.

FIG. 14 is a multi-column (panel) figure depicting experiment design,biochemical and clinical results in a series of non-human primates thatreceived intravenous nitrite to examine its effects on the developmentof vasospasm of the cerebral arteries and resulting ischemia. Each ofthe three columns represents a separate experimental group (control, lownitrite, and high nitrite). This figure describes experimental design(upper row: arrows pointing down marking the events; small arrowspointing up in the middle column representing daily boluses of nitrite),biochemical results (linear graphs: red, nitrite levels in blood; blue,nitrite levels in CSF; green, levels of nitrosylated protein/albumin inCSF; the brown bar graph represents the methemoglobin levels in blood),and mean blood pressure (the last grey bar graph) in samples collectedduring the experiment.

FIGS. 15A-15D present characteristic cerebral arteriograms before SAH(Day 0 (preinfusion); FIG. 15A, 15C) and on day 7 after SAH (FIG. 15B,15D) in two animals: one control treated with intravenous infusion ofsaline at 2 μl/min for 14 days (FIG. 15A, 15B) and one treated withintravenous nitrite at 870 μmol/min for 14 days (FIG. 15C, 15D). In FIG.15B, the arrows point to the right middle cerebral artery (R MCA) inspasm. R ICA, the right internal carotid artery, R ACA, the rightanterior cerebral artery.

FIG. 16 depicts degree of vasospasm of the right middle cerebral artery(R MCA) in each animal from all experimental groups (8 control, 3 lowdose, and 3 high dose of nitrite). R MCA vasospasm was assessed as thearea of the proximal 14-mm segment of the right MCA by three blindedexaminers using a computerized image analysis system (NIH Image 6.21).Arteriographic vasospasm was quantified relative to each animal baselinearteriogram. The mean values for saline vs. nitrite groups arerepresented by the circles; bars represent standard deviations.Statistical significance p<0.001.

DETAILED DESCRIPTION OF THE DISCLOSURE

I. Abbreviations

-   -   ANOVA analysis of variance    -   carboxy-PTIO        2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide        potassium salt    -   DCV delayed cerebral vasospasm    -   deoxy-RBC deoxygenated red blood cells    -   eNOS endothelial NO synthase    -   FiO₂ fractional concentration of inspired oxygen    -   FBF forearm blood flow    -   iNO inhaled nitric oxide    -   I/R ischemia-reperfusion    -   LCA main coronary artery    -   L-NMMA L-NG-monomethyl-arginine    -   LV left ventricle    -   NO nitric oxide    -   NOS nitric oxide synthase    -   MAP mean arterial pressure    -   MetHb methemoglobin    -   oxy-RBC oxygenated red blood cells    -   PBS phosphate buffered saline    -   pO₂ (or Po₂) partial oxygen pressure    -   SAH subarachnoid hemorrhage    -   S-NO S-nitroso-hemoglobin        II. Terms

Unless otherwise noted, terms used herein should be accorded theirstandard definitions and conventional usage. For example, one of skillin the art can obtain definitions for the terms used herein indictionaries and reference textbooks, for example: Stedman's MedicalDictionary (26^(th) Ed., Williams and Wilkins, Editor M. Spraycar,1995); The New Oxford American Dictionary (Oxford University Press, EdsE. Jewell and F. Abate, 2001); Molecular Cloning: A Laboratory Manual(Sambrook et al., 3^(rd) Ed., Cold Spring Harbor Laboratory Press,2001); and Hawley's Condensed Chemical Dictionary, 11^(th) Ed. (Eds. N.I. Sax and R. J. Lewis, Sr., Van Nostrand Reinhold, New York, N.Y.,1987); Molecular Biology and Biotechnology: a Comprehensive DeskReference (VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8)).

In order to facilitate review of the various embodiments, the followingexplanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals.

Cerebral ischemia or ischemic stroke: A condition that occurs when anartery to or in the brain is partially or completely blocked such thatthe oxygen demand of the tissue exceeds the oxygen supplied. Deprived ofoxygen and other nutrients following an ischemic stroke, the brainsuffers damage as a result of the stroke.

Ischemic stroke can be caused by several different kinds of diseases.The most common problem is narrowing of the arteries in the neck orhead. This is most often caused by atherosclerosis, or gradualcholesterol deposition. If the arteries become too narrow, blood cellsmay collect in them and form blood clots (thrombi). These blood clotscan block the artery where they are formed (thrombosis), or can dislodgeand become trapped in arteries closer to the brain (embolism).

Another cause of stroke is blood clots in the heart, which can occur asa result of irregular heartbeat (for example, atrial fibrillation),heart attack, or abnormalities of the heart valves. While these are themost common causes of ischemic stroke, there are many other possiblecauses. Examples include use of street drugs, traumatic injury to theblood vessels of the neck, or disorders of blood clotting.

Ischemic stroke is by far the most common kind of stroke, accounting forabout 80% of all strokes. Stroke can affect people of all ages,including children. Many people with ischemic strokes are older (60 ormore years old), and the risk of stroke increases with older ages. Ateach age, stroke is more common in men than women, and it is more commonamong African-Americans than white Americans. Many people with strokehave other problems or conditions which put them at higher risk forstroke, such as high blood pressure (hypertension), heart disease,smoking, or diabetes.

Fetal: A term describing the time period in the latter part of pregnancywhen organ systems are functional and blood flow patterns areestablished for central critical organs, such as the heart, brain andlungs.

Hypoxia: Deficiency in the amount of oxygen reaching body tissues.

Injectable composition: A pharmaceutically acceptable fluid compositioncomprising at least one active ingredient, for example, a salt ofnitrite. The active ingredient is usually dissolved or suspended in aphysiologically acceptable carrier, and the composition can additionallycomprise minor amounts of one or more non-toxic auxiliary substances,such as emulsifying agents, preservatives, pH buffering agents and thelike. Such injectable compositions that are useful for use with thecompositions of this disclosure are conventional; appropriateformulations are well known in the art.

Ischemia: A vascular phenomenon in which a decrease in the blood supplyto a bodily organ, tissue, or part is caused, for instance, byconstriction or obstruction of one or more blood vessels. Ischemiasometimes results from vasoconstriction or thrombosis or embolism.Ischemia can lead to direct ischemic injury, tissue damage due to celldeath caused by reduced oxygen supply.

Ischemia/reperfusion injury: In addition to the immediate injury thatoccurs during deprivation of blood flow, ischemic/reperfusion injuryinvolves tissue injury that occurs after blood flow is restored. Currentunderstanding is that much of this injury is caused by chemical productsand free radicals released into the ischemic tissues.

When a tissue is subjected to ischemia, a sequence of chemical events isinitiated that may ultimately lead to cellular dysfunction and necrosis.If ischemia is ended by the restoration of blood flow, a second seriesof injurious events ensue producing additional injury. Thus, wheneverthere is a transient decrease or interruption of blood flow in asubject, the resultant injury involves two components—the direct injuryoccurring during the ischemic interval and the indirect or reperfusioninjury that follows. When there is a long duration of ischemia, thedirect ischemic damage, resulting from hypoxia, is predominant. Forrelatively short duration ischemia, the indirect or reperfusion mediateddamage becomes increasingly important. In some instances, the injuryproduced by reperfusion can be more severe than the injury induced byischemia per se. This pattern of relative contribution of injury fromdirect and indirect mechanisms has been shown to occur in all organs.

Methemoglobin: The oxidized form of hemoglobin in which the iron in theheme component has been oxidized from the ferrous (+2) to the ferric(+3) state. This renders the hemoglobin molecule incapable ofeffectively transporting and releasing oxygen to the tissues. Normally,there is about 1% of total hemoglobin in the methemoglobin form.

Methemoglobinemia: A condition in which a substantial portion of thehemoglobin in the blood of a subject is in the form of methemoglobin,making it unable to carry oxygen effectively to the tissues.Methemoglobinemia can be an inherited disorder, but it also can beacquired through exposure to chemicals such as nitrates(nitrate-contaminated water), aniline dyes, and potassium chlorate. Itis not the presence of methemoglobin but the amount that is important inthe clinical setting. The following provides rough indications ofsymptoms associated with different levels of methemoglobin in the blood:<1.7%, normal; 10-20%, mild cyanosis (substantially asymptomatic, thoughit can result in “chocolate brown” blood); 30-40%, headache, fatigue,tachycardia, weakness, dizziness; >35%, symptoms of hypoxia, such asdyspnea and lethargy; 50-60%, acidosis, arrhythmias, coma, convulsions,bradycardia, severe hypoxia, seizures; >70% usually results in death.Neonate: A term describing the human or animal organism in the timeperiod after birth and extending until the adjustments from fetal tonewborn life are completed.

Nitrite: The inorganic anion ⁻NO₂ or a salt of nitrous acid (NO₂ ⁻).Nitrites are often highly soluble, and can be oxidized to form nitratesor reduced to form nitric oxide or ammonia. Nitrite may form salts withalkali metals, such as sodium (NaNO₂, also known as nitrous acid sodiumsalt), potassium and lithium, with alkali earth metals, such as calcium,magnesium and barium, with organic bases, such as amine bases, forexample, dicyclohexylamine, pyridine, arginine, lysine and the like.Other nitrite salts may be formed from a variety of organic andinorganic bases. In particular embodiments, the nitrite is a salt of ananionic nitrite delivered with a cation, which cation is selected fromsodium, potassium, and arginine. Many nitrite salts are commerciallyavailable, and/or readily produced using conventional techniques.

Parenteral: Administered outside of the intestine, for example, not viathe alimentary tract. Generally, parenteral formulations are those thatwill be administered through any possible mode except ingestion. Thisterm especially refers to injections, whether administeredintravenously, intrathecally, intramuscularly, intraperitoneally, orsubcutaneously, and various surface applications including intranasal,intradermal, and topical application, for instance.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Peripheral Vascular Disease (PVD): A condition in which the arteriesthat carry blood to the arms or legs become narrowed or occluded. Thisinterferes with the normal flow of blood, sometimes causing pain butoften causing no readily detectable symptoms at all.

The most common cause of PVD is atherosclerosis, a gradual process inwhich cholesterol and scar tissue build up, forming plaques that occludethe blood vessels. In some cases, PVD may be caused by blood clots thatlodge in the arteries and restrict blood flow. PVD affects about one in20 people over the age of 50, or 8 million people in the United States.More than half the people with PVD experience leg pain, numbness orother symptoms, but many people dismiss these signs as “a normal part ofaging” and do not seek medical help. The most common symptom of PVD ispainful cramping in the leg or hip, particularly when walking Thissymptom, also known as “claudication,” occurs when there is not enoughblood flowing to the leg muscles during exercise, such that ischemiaoccurs. The pain typically goes away when the muscles are rested.

Other symptoms may include numbness, tingling or weakness in the leg. Insevere cases, people with PVD may experience a burning or aching pain inan extremity such as the foot or toes while resting, or may develop asore on the leg or foot that does not heal. People with PVD also mayexperience a cooling or color change in the skin of the legs or feet, orloss of hair on the legs. In extreme cases, untreated PVD can lead togangrene, a serious condition that may require amputation of a leg, footor toes. People with PVD are also at higher risk for heart disease andstroke.

A “pharmaceutical agent” or “drug” refers to a chemical compound orother composition capable of inducing a desired therapeutic orprophylactic effect when properly administered to a subject.

Placenta: A vascular organ that provides for metabolic exchange betweenmother and fetus in mammals. It delivers oxygen, water, and nutrients tothe fetus from the mother's blood and secretes the hormones necessaryfor successful pregnancy. In addition, it carries wastes away from thefetus to be processed in the mother's body.

Preeclampsia: A disease of unknown cause in pregnant women,characterized by hypertension, abnormal blood vessels in the placenta,and protein in the urine. It often but not always occurs withgestational diabetes or in diabetics. Additional symptoms may includewater retention, leading to swelling in the face, hands and feet, andgreater weight gain. Also called toxemia. Preeclampsia can lead toeclampsia if not treated. The only known cure for preeclampsia isdelivery of the child.

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified nitritesalt preparation is one in which the specified nitrite salt is moreenriched than it is in its generative environment, for instance within abiochemical reaction chamber. Preferably, a preparation of a specifiednitrite salt is purified such that the salt represents at least 50% ofthe total nitrite content of the preparation. In some embodiments, apurified preparation contains at least 60%, at least 70%, at least 80%,at least 85%, at least 90%, at least 95% or more of the specifiedcompound, such as a particular nitrite salt.

Reperfusion: Restoration of blood supply to tissue that is ischemic, dueto decrease in blood supply. Reperfusion is a procedure for treatinginfarction or other ischemia, by enabling viable ischemic tissue torecover, thus limiting further necrosis. However, it is thought thatreperfusion can itself further damage the ischemic tissue, causingreperfusion injury.

Subject: Living multi-cellular organisms, including vertebrateorganisms, a category that includes both human and non-human mammals.

Therapeutic: A generic term that includes both diagnosis and treatment.

Therapeutically effective amount of [a vasodilator]: A quantity ofcompound, such as a nitrite salt, sufficient to achieve a desired effectin a subject being treated. For instance, this can be the amountnecessary to treat or ameliorate relatively high blood pressure, or tomeasurably decrease blood pressure over a period of time, or tomeasurably inhibit an increase in blood pressure, in a subject.

An effective amount of a vasodilator may be administered in a singledose, or in several doses, for example daily, during a course oftreatment. However, the effective amount will be dependent on thecompound applied, the subject being treated, the severity and type ofthe affliction, and the manner of administration of the compound. Forexample, a therapeutically effective amount of an active ingredient canbe measured as the concentration (moles per liter or molar-M) of theactive ingredient (such as a pharmaceutically-acceptable salt ofnitrite) in blood (in vivo) or a buffer (in vitro) that produces aneffect.

By way of example, as described herein it is now shown thatpharmaceutically-acceptable salts of nitrite (such as sodium nitrite)are effective as vasodilators at calculated dosages of about 0.6 toabout 200 μM final concentration of nitrite in the circulating blood ofa subject, which level can be determined empirically or throughcalculations. Specific levels can be reached, for instance, by providingless than about 200 mg or less nitrite in a single dose, or a doseprovided over a period of time (e.g., by infusion or inhalation). Forinstance, other dosages may be 150 mg, 100 mg, 75 mg, 50 mg or less.Specific example dosages of nitrite salts are provided herein, thoughthe examples are not intended to be limiting. Exact dosage amounts willvary by the size of the subject being treated, the duration of thetreatment, the mode of administration, and so forth.

Particularly beneficial therapeutically effective amounts of avasodilator, such as a pharmaceutically-acceptable nitrite salt (e.g.,sodium nitrite), are those that are effective for vasodilation orincreasing blood flow, but not so high that a significant or toxic levelof methemoglobin is produced in the subject to which the vasodilator isadministered. In specific embodiments, for instance, no more than about25% methemoglobin is produced in the subject. More preferably, no morethan 20%, no more than 15%, no more than 10%, no more than 8% or lessmethemoglobin is produced, for instance as little as 5% or 3% or less,in response to treatment with the vasodilator.

The compounds discussed herein have equal application in medical andveterinary settings. Therefore, the general term “subject being treated”is understood to include all animals (for example, humans, apes,laboratory animals, companion animals, etc.) that are or may besuffering from an aberration in blood pressure, such as hypertension.

Vasoconstriction. The diminution of the caliber or cross-sectional areaof a blood vessel, for instance constriction of arterioles leading todecreased blood flow to a body part. This can be caused by a specificvasoconstrictor, an agent (for instance a chemical or biochemicalcompound) that causes, directly or indirectly, constriction of bloodvessels. Such an agent can also be referred to as a vasohypertonicagent, and is said to have vasoconstrictive activity. A representativecategory of vasoconstrictors is the vasopressor (from the term pressor,tending to increase blood pressure), which term is generally used torefer to an agent that stimulates contraction of the muscular tissue ofthe capillaries and arteries.

Vasoconstriction also can be due to vasospasm, inadequatevasodilatation, thickening of the vessel wall, or the accumulation offlow-restricting materials on the internal wall surfaces or within thewall itself. Vasoconstriction is a major presumptive or proven factor inaging and in various clinical conditions including progressivegeneralized atherogenesis, myocardial infarction, stroke, hypertension,glaucoma, macular degeneration, migraine, hypertension and diabetesmellitus, among others.

Vasodilation. A state of increased caliber of the blood vessels, or theact of dilation of a blood vessel, for instance dilation of arteriolesleading to increased blood flow to a body part. This can be caused by aspecific vasodilator, an agent (for instance, a chemical or biochemicalcompound) that causes, directly or indirectly, dilation of bloodvessels. Such an agent can also be referred to as a vasohypotonic agent,and is said to have vasodilative activity.

Vasospasm: Another cause of stroke occurs secondary to spasm of bloodvessels supplying the brain. This type of stroke typically follows asubarachnoid aneurismal hemorrhage with a delayed development ofvasospasm within 2-3 weeks of the bleeding event. A similar type ofstroke may complicate sickle cell disease.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview of Several Embodiments

It has been surprisingly discovered that administration ofpharmaceutically-acceptable salts of nitrite is useful in the regulationof the cardiovascular system. It has also been surprisingly discoveredthat nitrite is reduced to nitric oxide in vivo, and that the nitricoxide produced thereby is an effective vasodilator. These effectssurprisingly occur at doses that do not produce clinically significantmethemoglobinemia. These discoveries now enable methods to prevent andtreat conditions associated with the cardiovascular system, for example,high blood pressure, pulmonary hypertension, cerebral vasospasm andtissue ischemia-reperfusion injury. These discoveries also providemethods to increase blood flow to tissues, for example, to tissues inregions of low oxygen tension. It is particularly surprising that thenitrite does not need to be applied in an acidified condition in orderfor it to be effective in regulating the cardiovascular system, and moreparticularly to act as a vasodilator in vivo.

Accordingly, the present disclosure provides in one embodiment a methodfor decreasing a subject's blood pressure, including administering tothe subject sodium nitrite at about 36 μmoles per minute or less intothe forearm brachial artery or intravenously.

The present disclosure also provides a method for decreasing a subject'sblood pressure, including administering to the subject an effectiveamount of pharmaceutically-acceptable nitrite so as to decrease (orlower, or reduce) the subject's blood pressure. Another embodiment is amethod for treating a subject having a condition associated withelevated blood pressure, including administering to the subject aneffective amount of pharmaceutically-acceptable nitrite so as to treatat least one vascular complication associated with the elevated bloodpressure. Also provided is a method for treating a subject having ahemolytic condition, including administering to the subject an effectiveamount of pharmaceutically-acceptable nitrite so as to treat at leastone vascular complication associated with the hemolytic condition.

The present disclosure additionally provides a method for increasingblood flow to a tissue of a subject, including administering to thesubject an effective amount of pharmaceutically-acceptable nitrite so asto increase blood flow to a tissue of the subject. Also provided is amethod for producing an amount of NO in a subject effective the decreasethe subject's blood pressure, including administering apharmaceutically-acceptable nitrite to the subject.

The present disclosure further provides a pharmaceutical compositioncomprising an effective amount of a pharmaceutically-acceptable nitriteand a carrier.

In some embodiments, the vascular complication is one or more selectedfrom the group consisting of pulmonary hypertension (including neonatalpulmonary hypertension, primary pulmonary hypertension, and secondarypulmonary hypertension), systemic hypertension, cutaneous ulceration,acute renal failure, chronic renal failure, intravascular thrombosis, anischemic central nervous system event, and death.

In some embodiments, nitrite is administered to neonates to treatpulmonary hypertension.

In some embodiments, the hemolytic condition includes one or moreselected from: sickle cell anemia, thalassemia, hemoglobin C disease,hemoglobin SC disease, sickle thalassemia, hereditary spherocytosis,hereditary elliptocytosis, hereditary ovalcytosis, glucose-6-phosphatedeficiency and other red blood cell enzyme deficiencies, paroxysmalnocturnal hemoglobinuria (PNH), paroxysmal cold hemoglobinuria (PCH),thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS),idiopathic autoimmune hemolytic anemia, drug-induced immune hemolyticanemia, secondary immune hemolytic anemia, non-immune hemolytic anemiacaused by chemical or physical agents, malaria, falciparum malaria,bartonellosis, babesiosis, clostridial infection, severe Haemophilusinfluenzae type b infection, extensive burns, transfusion reaction,rhabdomyolysis (myoglobinemia), transfusion of aged blood,cardiopulmonary bypass, and hemodialysis.

In some embodiments, the decreased blood flow to the tissue is causeddirectly or indirectly by at least one of the following conditions:sickle cell anemia, thalassemia, hemoglobin C disease, hemoglobin SCdisease, sickle thalassemia, hereditary spherocytosis, hereditaryelliptocytosis, hereditary ovalcytosis, glucose-6-phosphate deficiencyand other red blood cell enzyme deficiencies, paroxysmal nocturnalhemoglobinuria (PNH), paroxysmal cold hemoglobinuria (PCH), thromboticthrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), idiopathicautoimmune hemolytic anemia, drug-induced immune hemolytic anemia,secondary immune hemolytic anemia, non-immune hemolytic anemia caused bychemical or physical agents, malaria, falciparum malaria, bartonellosis,babesiosis, clostridial infection, severe Haemophilus influenzae type binfection, extensive burns, transfusion reaction, rhabdomyolysis(myoglobinemia), transfusion of aged blood, transfusion of hemoglobin,transfusion of red blood cells, cardiopulmonary bypass, coronarydisease, cardiac ischemia syndrome, angina, iatrogenic hemolysis,angioplasty, myocardial ischemia, tissue ischemia, hemolysis caused byintravascular devices, hemodialysis, pulmonary hypertension, systemichypertension, cutaneous ulceration, acute renal failure, chronic renalfailure, intravascular thrombosis, and an ischemic central nervoussystem event.

In some embodiments, the tissue is an ischemic tissue. In someembodiments, the administration is parenteral, oral, buccal, rectal, exvivo, or intraocular. In some embodiments, the administration isperitoneal, intravenous, intraarterial, subcutaneous, inhaled, orintramuscular. In some embodiments, the nitrite is administered to thesubject in an environment of low oxygen tension, or acts in an area ofthe subject's body that displays relatively low oxygen tension. In someembodiments, the nitrite is administered as apharmaceutically-acceptable salt of nitrite, such as, for instance,sodium nitrite, potassium nitrite, or arginine nitrite. In someembodiments, the nitrite is administered in combination with at leastone additional active agent. It is specifically contemplated that, incertain embodiments, that the subject is a mammal, for instance, ahuman.

The disclosure further provides a method for treating a subject having acondition associated with elevated blood pressure in the lungs, e.g.pulmonary hypertension, including administering to the subject aneffective amount of pharmaceutically-acceptable nitrite. In someembodiments, this includes treating a subject having neonatal pulmonaryhypertension. In some embodiments, this includes treating a subjecthaving primary and/or secondary pulmonary hypertension. In someembodiments for treating subjects having a condition associated withelevated blood pressure in the lungs, the nitrite is nebulized.

The disclosure also provides suggestions for a means of treatinghypertension and/or preeclampsia in pregnant women. Such therapy wouldinclude action of nitrites on spastic and diseased blood vessels withinthe placenta.

The disclosure also provides suggestions for treating, in utero, fetuseswith cardiovascular anomalies, hypertension, and/or misdirected bloodflow. In such approaches, nitrite may be administered by introductioninto the amniotic cavity either directly or by osmotic minipumps, thelatter to achieve sustained release throughout days and weeks ofpregnancy.

Thus, there is provided herein a method for inducing vasodilation and/orincreasing blood flow in a subject, which method involves administeringto the subject an effective amount of a pharmaceutically-acceptable saltof nitrite for a sufficient period of time to induce vasodilation and/orincrease blood flow in the subject. Non-limiting examples ofpharmaceutically acceptable salts of nitrite include sodium nitrite,potassium nitrite, and arginine nitrite. In examples of the providedmethods, the pharmaceutically-acceptable salt of nitrite reacts in thepresence of hemoglobin in the subject to release nitric oxide.

It is a specific advantage of methods provided herein that the effectiveamount of the pharmaceutically-acceptable salt of nitrite administeredto the subject does not induce toxic levels of methemoglobin, and inmany embodiments does not induced formation of clinically significantamounts of methemoglobin in the subject. Therefore, contemplated hereinare methods in which the effective amount of thepharmaceutically-acceptable salt of nitrite, when administered to thesubject, induces production in the subject of no more than about 25%methemoglobin; no more than about 20% methemoglobin; no more than about10% methemoglobin; no more than about 8% methemoglobin; or no more thanabout 5% methemoglobin. Beneficially, examples of the provided methodsinduce production of even less than 5% methemoglobin, for instance nomore than about 3% methemoglobin, less than 3%, less than 2%, or evenless than 1%.

In one specific example of a method for inducing vasodilation and/orincreasing blood flow in a subject, sodium nitrite is administered byinjection at about 36 μmoles per minute for at least five minutes intothe forearm brachial artery of the subject.

The effective amount of the pharmaceutically-acceptable salt of nitriteis administered, in various embodiments, to a circulating concentrationin the subject of about 0.6 to 240 μM, measured locally to the site ofadministration or generally in the subject. It is noted that the locallevel of nitrite is expected to be higher than the general circulatinglevel particularly in short delivery regimens; in long term deliveryregimens, such as delivery using a pump or injector, or by inhalation,the system-wide or general nitrite level is expected to near the levelmeasured near the administration site.

Administration of the pharmaceutically-acceptable nitrite can be, forinstance, parenteral, oral, buccal, rectal, ex vivo, or intraocular incertain embodiments. In various embodiments, it is also contemplatedthat the administration of the nitrite can be peritoneal, intravenous,intraarterial, subcutaneous, inhaled, intramuscular, or into acardiopulmonary bypass circuit. Combinations of two or more routes ofadministration are also contemplated.

In various embodiments of the method for inducing vasodilation and/orincreasing blood flow in a subject, the subject is a mammal. It isparticularly contemplated that the subject can be a human.

Combination therapy methods are contemplated, wherein the nitrite isadministered in combination with at least one additional agent. By wayof non-limiting examples, the additional agent is one or more selectedfrom the list consisting of penicillin, hydroxyurea, butyrate,clotrimazole, arginine, or a phosphodiesterase inhibitor (such assildenafil).

In another embodiment of the method for inducing vasodilation and/orincreasing blood flow in a subject, the subject has elevated bloodpressure, and the method is a method for treating at least one vascularcomplication associated with the elevated blood pressure, or the subjecthas a hemolytic condition, and the method is a method for treating atleast one vascular complication associated with the hemolytic condition.Optionally, the subject may have both elevated blood pressure and ahemolytic condition.

In examples of the methods provided herein, the at least one vascularcomplication is one or more selected from the group consisting ofpulmonary hypertension, systemic hypertension, peripheral vasculardisease, trauma, cardiac arrest, general surgery, organ transplantation,cutaneous ulceration, acute renal failure, chronic renal failure,intravascular thrombosis, angina, an ischemia-reperfusion event, anischemic central nervous system event, and death.

In examples of the methods in which the subject has a hemolyticcondition, the hemolytic condition is one or more selected from thegroup consisting of sickle cell anemia, thalassemia, hemoglobin Cdisease, hemoglobin SC disease, sickle thalassemia, hereditaryspherocytosis, hereditary elliptocytosis, hereditary ovalcytosis,glucose-6-phosphate deficiency and other red blood cell enzymedeficiencies, paroxysmal nocturnal hemoglobinuria (PNH), paroxysmal coldhemoglobinuria (PCH), thrombotic thrombocytopenic purpura/hemolyticuremic syndrome (TTP/HUS), idiopathic autoimmune hemolytic anemia,drug-induced immune hemolytic anemia, secondary immune hemolytic anemia,non-immune hemolytic anemia caused by chemical or physical agents,malaria, falciparum malaria, bartonellosis, babesiosis, clostridialinfection, severe Haemophilus influenzae type b infection, extensiveburns, transfusion reaction, rhabdomyolysis (myoglobinemia), transfusionof aged blood, transfusion of hemoglobin, transfusion of red bloodcells, cardiopulmonary bypass, coronary disease, cardiac ischemiasyndrome, angina, iatrogenic hemolysis, angioplasty, myocardialischemia, tissue ischemia, hemolysis caused by intravascular devices,and hemodialysis.

In yet another embodiment of the method for inducing vasodilation and/orincreasing blood flow in a subject, the subject has a conditionassociated with decreased blood flow to a tissue, and the method is amethod to increase blood flow to the tissue of the subject. Forinstance, in examples of this method, the decreased blood flow to thetissue is caused directly or indirectly by at least one conditionselected from the group consisting of: sickle cell anemia, thalassemia,hemoglobin C disease, hemoglobin SC disease, sickle thalassemia,hereditary spherocytosis, hereditary elliptocytosis, hereditaryovalcytosis, glucose-6-phosphate deficiency and other red blood cellenzyme deficiencies, paroxysmal nocturnal hemoglobinuria (PNH),paroxysmal cold hemoglobinuria (PCH), thrombotic thrombocytopenicpurpura/hemolytic uremic syndrome (TTP/HUS), idiopathic autoimmunehemolytic anemia, drug-induced immune hemolytic anemia, secondary immunehemolytic anemia, non-immune hemolytic anemia caused by chemical orphysical agents, malaria, falciparum malaria, bartonellosis, babesiosis,clostridial infection, severe Haemophilus influenzae type b infection,extensive burns, transfusion reaction, rhabdomyolysis (myoglobinemia),transfusion of aged blood, transfusion of hemoglobin, transfusion of redblood cells, cardiopulmonary bypass, coronary disease, cardiac ischemiasyndrome, angina, iatrogenic hemolysis, angioplasty, myocardialischemia, tissue ischemia, hemolysis caused by intravascular devices,hemodialysis, pulmonary hypertension, systemic hypertension, cutaneousulceration, acute renal failure, chronic renal failure, intravascularthrombosis, and an ischemic central nervous system event.

It is specifically contemplated in examples of this method that thetissue is an ischemic tissue, for instance one or more tissues selectedfrom the group consisting of neuronal tissue, bowel tissue, intestinaltissue, limb tissue, lung tissue, central nervous tissue, or cardiactissue.

Also provided are methods for inducing vasodilation and/or increasingblood flow in a subject having elevated blood pressure, wherein theelevated blood pressure comprises elevated blood pressure in the lungs.By way of example, it is contemplated that such subject in someinstances has neonatal pulmonary hypertension, or primary and/orsecondary pulmonary hypertension.

In examples of embodiments where the elevated blood pressure, or needfor increased blood flow, in the subject comprises elevated bloodpressure or need for increased blood flow in the lungs, thepharmaceutically-acceptable salt of nitrite is nebulized.

By way of example, in various embodiments thepharmaceutically-acceptable salt of nitrite is administered to acirculating concentration in the subject of no more than about 100 μM;no more than about 50 μM; no more than about 20 μM; no more than about16 μM; or less than about 16 μM.

Another embodiment is a method for treating or ameliorating a conditionselected from: (a) hepatic or cardiac or brain ischemia-reperfusioninjury; (b) pulmonary hypertension; or (c) cerebral artery vasospasm, ina subject by decreasing blood pressure and/or increasing vasodilation inthe subject, the method comprising administering sodium nitrite to thesubject to decrease the blood pressure and/or increase vasodilation inthe subject, thereby treating or ameliorating the condition.

In specific examples of this embodiment, the method is a method fortreating or ameliorating hepatic or cardiac or brainischemia-reperfusion injury. Optionally, the sodium nitrite isadministered to the subject via injection, for instance, intravenousinjection. In certain examples, the sodium nitrite is administered to acirculating concentration of about 0.6 to 240 μM.

In other specific examples of this embodiment, the method is a methodfor treating or ameliorating pulmonary hypertension, such as forinstance neonatal pulmonary hypertension. Beneficially, in such methodsthe sodium nitrite can be administered to the subject by inhalation, forinstance it can be nebulized. Optionally, in any of these methods, thesodium nitrite is administered at a rate of 270 μmol/minute, thoughother rates and circulating levels are contemplated.

Also provided in other examples of this embodiment are methods fortreating or ameliorating cerebral artery vasospasm. Optionally, thesodium nitrite is administered to the subject via injection, forinstance, intravenous injection. In examples of such methods, the sodiumnitrite is administered at a rate of about 45 to 60 mg/kg.

In examples of the described methods, optionally the sodium nitrite canbe administered in combination with at least one additional agent.

In any of the described methods, it is contemplated that the subject canbe a mammal, such as for instance a human.

IV. Sodium Nitrite as an In Vivo Vasodilator

Nitrite anions are present in concentrations of about 150-1000 nM in theplasma and about 10 μM in aortic tissue. This represents the largestvascular storage pool of nitric oxide (NO), provided physiologicalmechanisms exist to reduce nitrite to NO. The vasodilator properties ofnitrite in the human forearm and the mechanisms extant for itsbioactivation have been investigated and results are reported herein.Sodium nitrite was infused at about 36 μmoles per minute into theforearm brachial artery of 18 normal volunteers, resulting in a regionalnitrite concentration of about 222 μM and an immediate about 175%increase in resting forearm blood flow. Increased blood flow wasobserved at rest, during NO synthase inhibition and with exercise, andresulted in increased tissue perfusion, as demonstrated by increases invenous hemoglobin-oxygen saturation, partial pressure of oxygen, and pH.Systemic concentrations of nitrite increased to about 16 μM andsignificantly reduced mean arterial blood pressure. In an additional sixsubjects, the dose of nitrite was reduced about 2-logs and infused at360 nmoles per minute, resulting in a forearm nitrite concentration ofabout 2 μM and an about 22% increase in blood flow.

Nitrite infusions were associated with the formation of erythrocyteiron-nitrosyl-hemoglobin, and to a lesser extent, S-nitroso-hemoglobinacross the forearm vasculature. The formation of NO-modified hemoglobinappears to result from the nitrite reductase activity ofdeoxyhemoglobin, linking tissue hypoxia and nitrite bioactivation.

These results indicate that physiological levels of blood and tissuenitrite represent a major bioavailable pool of NO that contributes tovaso-regulation and provides a mechanism for hypoxic vasodilation viareaction of vascular nitrite with deoxygenated heme proteins.Substantial blood flow effects of nitrite infusion into the brachialartery of normal human subjects results from forearm nitriteconcentrations as low as about 0.9 μM.

By way of example, as described herein it is now shown thatpharmaceutically-acceptable salts of nitrite (such as sodium nitrite)are effective as vasodilators at calculated dosages of about 0.6 toabout 200 μM final concentration of nitrite in the circulating blood ofa subject. Specific circulating levels (locally or generally in thesubject) can be reached, for instance, by providing less than about 200mg or less nitrite in a single dose, or a dose provided over a period oftime (e.g., by infusion or inhalation). For instance, other dosages maybe 150 mg, 100 mg, 75 mg, 50 mg or less. Specific example dosages ofnitrite salts are provided herein, though the examples are not intendedto be limiting. Exact dosage amounts will vary by the size of thesubject being treated, the duration of the treatment, the mode ofadministration, and so forth.

Infusion rates can be calculated, for any given desired targetcirculating concentration, by using the following equation:Infusion rate (μM/min)=target concentration (μmol/L, or μM)×Clearance(L/min)

where Clearance (L/min)=0.015922087×weight of the subject (kg)↑0.8354

The rate of clearance has been calculated based on empirical results,including those reported herein.

By way of example, when sodium nitrite is infused into a human forearmat 36 micromoles (μMol) per minute, the concentration measured comingout of forearm is about 222 μM and about 16 μM in whole body, after 15minutes infusion. The background level of circulating nitrite in mammalsis low, around 150-500 nanoM.

Particularly beneficial therapeutically effective amounts of avasodilator, such as a pharmaceutically-acceptable nitrite salt (e.g.,sodium nitrite), are those that are effective for vasodilation orincreasing blood flow, but not so high that a significant or toxic levelof methemoglobin is produced in the subject to which the vasodilator isadministered. In specific embodiments, for instance, no more than about25% methemoglobin is produced in the subject. More preferably, no morethan 20%, no more than 15%, no more than 10%, no more than 8% or lessmethemoglobin is produced, for instance as little as 5% or 3% or less,in response to treatment with the vasodilator.

By way of specific example, nitrite can be infused at concentrationsless than 40 μMol per minute intravenously or intraarterially, or givenby mouth. Importantly, doses used are less than those used for thetreatment of cyanide poisoning, which are designed to induce clinicallysignificant methemoglobinemia. Surprisingly, the doses described hereinfor the treatment/prevention of cardiovascular conditions producesignificant and beneficial clinical effects without clinicallysignificant methemoglobin production.

Relatively complex inorganic/organic nitrite compounds and nitratecompounds have been utilized clinically to treat disorders, includingangina. These drugs (e.g., glyceryl trinitrate) suffer from tolerance(requiring increases in dosage in order to maintain the same effect),however, and are distinct vasodilators compared to nitrite. For example,the former require cellular thiols for metabolism, whereas nitrite orthe nitrite salts discussed herein (e.g., sodium nitrite) do not.

V. A Mechanism of Iron-nitrosyl- and S-nitroso-hemoglobin Formation InVivo

The levels of both iron-nitrosyl- and S-nitroso-hemoglobin formed invivo in this study are striking During a transit time of less than 10seconds through the forearm circulation during exercise, infused nitrite(200 μM regional concentration) produced approximately 750 nMiron-nitrosyl-hemoglobin and 200 nM SNO-Hb. The formation of bothNO-hemoglobin adducts was inversely correlated with hemoglobin-oxygensaturation, which fell during exercise stress, measured from theantecubital vein by co-oximetry (for iron-nitrosyl-hemoglobin r=−0.7,P<0.0001; for S-nitroso-hemoglobin r=−0.45, P=0.04; FIG. 4B). Additionof 200 μM nitrite to whole blood at different oxygen tensions (0-100%)recapitulated the in vivo data with increasing concentrations ofiron-nitrosyl hemoglobin being formed at lower oxygen tensions (foriron-nitrosyl-hemoglobin r=−0.968, P<0.0001; for S-nitroso-hemoglobinr=−0.45, P=0.07), strongly suggesting that the NO and SNO formation wasdependent on the reaction of nitrite with deoxyhemoglobin.

These data are consistent with the reaction of nitrite withdeoxyhemoglobin to form NO and iron-nitrosyl-hemoglobin (Doyle et al., JBiol Chem, 256, 12393-12398, 1981). Nitrite is first reduced to form NOand methemoglobin with a rate constant of 2.9 M⁻¹sec⁻¹ (measured at 25°C., pH 7.0). This reaction will be pseudo-first order, governed by theamounts (20 mM) of intra-erythrocytic hemoglobin, and limited by therate of nitrite uptake by the erythrocyte membrane. NO then binds todeoxyhemoglobin to form iron-nitrosyl-hemoglobin, escapes theerythrocyte, or reacts with other higher oxides, such as NO₂, to formN₂O₃ and S-nitroso-hemoglobin.

Equation Series 1NO₂ ⁻(nitrite)+HbFe^(II) (deoxyhemoglobin)+H⁺→HbFe^(III)(methemoglobin)+NO+OH⁻NO+HbFe^(II) (deoxyhemoglobin)→HbFe^(II)NO (iron-nitrosyl-hemoglobin)

The formation of significant amounts of S-nitroso-hemoglobin in vivoduring nitrite infusion was also observed. Luschinger and colleagues(Proc Natl Acad Sci USA, 100, 461-6, 2003) recently proposed thatnitrite reacts with deoxyhemoglobin to make iron-nitrosyl-hemoglobin,with subsequent “transfer” of the NO to the cysteine 93 to formS-nitroso-hemoglobin mediated by reoxygenation and quaternary T to Rtransition of hemoglobin. However, a direct transfer of NO from the hemeto the thiol requires NO oxidation to NO+ and such “cycling” has notbeen reproduced by other research groups. Fernandez and colleagues haverecently suggested that nitrite catalyzes the reductive nitrosylation ofmethemoglobin by NO, a process that generates the intermediatenitrosating species dinitrogen teraoxide (N₂O₃) (Inorg Chem, 42, 2-4,2003). However, nitrite reactions with hemoglobin provide idealconditions for NO and S-nitrosothiol generation along the oxygengradient as nitrite reacts with deoxyhemoglobin to form NO and withoxyhemoglobin to form nitrogen dioxide (NO₂) radical. NO₂ participatesin radical-radical reactions (k=10⁹ M⁻¹ sec⁻¹) with NO to form N₂O₃ andS-nitrosothiol. Additional chemistry of nitrite with hemoglobin producesreactive oxygen metabolites (such as superoxide and hydrogen peroxide;Watanabe et al., Acta Med Okayama 35, 173-8, 1981; Kosaka et al.,Biochim Biophys Acta 702, 237-41, 1982; and Kosaka et al., EnvironHealth Perspect 73, 147-51, 1987). Chemistry involving such NOradical-oxygen radical reactions provides competitive pathways forS-nitrosothiol formation in the presence of high affinity NO sinks, suchas hemoglobin.

VI. Physiological Considerations

The last decade has seen an increase in the understanding of thecritical role nitric oxide (NO) plays in vascular homeostasis. Thebalance between production of NO and scavenging of NO determines NObioavailability, and this balance is carefully maintained in normalphysiology. The homeostatic, vasoregulatory system is apparentlyfine-tuned to scavenge excess NO to limit gross endocrine actions whileallowing for sufficient local NO necessary for regional tonicvasodilation. However, rapid NO scavenging by cell-free hemoglobindisrupts this balance (Reiter et al., Nat Med 8, 1383-1389, 2002). Undernormal physiological conditions, hemoglobin is rapidly and effectivelycleared by the hemoglobin scavenger system. However, chronic hemolyticconditions, such as sickle cell disease, result in the daily release ofsubstantial quantities of hemoglobin into the vasculature, suggestingthat cell-free hemoglobin may have major systemic effects on NObioavailability. A current focus of research attempts to explain andtreat the vascular complications common to many chronic hemolyticconditions, such as pulmonary hypertension, cutaneous ulceration andacute and chronic renal failure. Similarly, a number of clinicaldiseases and therapies such as acute hemolytic crises, hemolysis duringcardiopulmonary bypass procedures, transfusion of aged blood, andmyoglobinuria following muscle infarction are often complicated by acutepulmonary and systemic hypertension, acute renal failure, intravascularthrombosis, ischemic central nervous system events and/or death.

It is demonstrated herein that nitrite produces vasodilation in humansassociated with nitrite reduction to NO by deoxyhemoglobin. Remarkably,systemic levels of 16 μM resulted in systemic vasodilation and decreasedblood pressure, and regional forearm levels of only 1-2 μM significantlyincreased blood flow at rest and with exercise stress. Furthermore,conversion of nitrite to NO and S-nitrosothiol was mediated by reactionwith deoxyhemoglobin, providing a mechanism for hypoxia-regulatedcatalytic NO production by the erythrocyte or endothelial/tissue hemeproteins. While high concentrations of hemoglobin in red cells, coupledwith the near diffusion-limited reaction rates (˜10⁷ M⁻¹s⁻¹) of NO withhemoglobin, seem to prohibit NO from being exported from the red bloodcell, the data presented herein argue to the contrary. While notintending to be limiting, perhaps unique characteristics of theerythrocyte membrane, with a submembrane protein and methemoglobin-richmicroenvironment, and the relative lipophilic nature of NO, allowcompartmentalized NO production at the red blood cell membrane. This,coupled with the small yields of NO necessary for vasodilation, couldaccount for the export of NO despite these kinetic constraints. It isfurther proposed that in vivo chemistry for the conversion of nitrite toNO and S-nitrosothiol by reaction with deoxyhemoglobin and methemoglobinprovides a mechanism for hypoxia-regulated catalytic NO production bythe erythrocyte or endothelial tissue heme proteins.

Three factors uniquely position nitrite, rather than S-nitrosothiol, asthe major vascular storage pool of NO: 1) Nitrite is present insubstantial concentrations in plasma, erythrocytes and in tissues(Rodriguez et al., Proc Natl Acad Sci USA 100:336-341, 2003). 2) Nitriteis relatively stable, because it is not readily reduced by intracellularreductants, as are S-nitrosothiols (Gladwin et al., J Biol Chem 21:21,2002) and its reaction rate with heme proteins is 10,000 times less thanthat of authentic NO. 3) Nitrite is only converted to NO by reactionwith deoxyhemoglobin (or presumably deoxy-myoglobin, -cytoglobin, and-neuroglobin) and its “leaving group” is the met(ferric)heme proteinwhich will not scavenge and inactivate NO (Doyle et al., J Biol Chem256:12393-12398, 1981). Therefore, this pool provides the idealsubstrate for NO generation during hypoxia, providing a novel mechanismfor hypoxic vasodilation.

Because a deoxyhemoglobin-nitrite reductase system would result in NOformation in deoxygenating blood, such a system links hemoglobinoxygenation status to NO generation, the principle previously ascribedto S-nitroso-hemoglobin (Jia et al., Nature 380:221-226, 1996).Hemoglobin possesses anionic binding cavities that retain nitrite(Gladwin et al., J Biol Chem 21:21, 2002) and nitrite is taken up byerythrocytes through the anion exchange protein (AE1 or Band 3) orthrough the membrane as nitrous acid (a pH dependent process thataccelerates nitrite uptake during tissue hypoxia (Shingles et al., JBioenerg Biomembr 29:611-616, 1997; May et al., Am I Physiol CellPhysiol 279:C1946-1954, 2000). Such nitrite would provide a steadysource of NO, NO₂ and S-nitrosothiol generation that would occurpreferentially in hypoxic vascular territories. Because the AE1 proteinbinds both deoxyhemoglobin and methemoglobin and may channel nitrite,AE1 could serve to localize catalytic NO and S-nitrosothiol generationat the erythrocyte membrane, where the relatively lipophilic NO, NO₂ andN₂O₃ could react in the vicinal lipid bilayer (FIG. 5). The erythrocytemembrane is lined by an unstirred outer diffusion barrier and an innermethemoglobin rich protein matrix that might further promote such NO andNO₂ chemistry (Coin et al., J Biol Chem 254:1178-1190, 1979; Liu et al.,J Biol Chem 273:18709-18713, 1998; Han et al., Proc Natl Acad Sci USA99:7763-7768, 2002).

This model is consistent with the in vitro observations of Pawloski andcolleagues (Pawloski et al., Nature 409:622-626, 2001) showing thatS-nitrosation of hemoglobin and AE1 occurs in the erythrocyte membraneafter treatment of deoxygenated red blood cells with NO solutions (whichcontain significant-more than 50 μM-contaminating nitrite; Fernandez, etal. Inorg Chem 42:2-4, 2003). Further, N₂O₃ generated at the membranecould directly nitrosate the abundant intra-erythrocytic glutathione,eliminating the requirement of transnitrosation reactions withS-nitroso-hemoglobin and thus facilitating rapid export of low molecularweight S-nitrosothiol by simple diffusion across the erythrocytemembrane (FIG. 5). A nitrite-hemoglobin chemistry supports a role forthe red cell in oxygen-dependent NO homeostasis and provides a mechanismfor the observations of multiple research groups that red blood cellsand plasma “loaded” with NO, by exposure to NO in high concentration insolution or to NO gas or donors (in equilibria with high concentrationsof nitrite), can export NO and induce vasodilation in vitro and in vivo(Rassaf et al., J Clin Invest 109:1241-1248, 2002; Fox-Robichaud et al.,J Glitz Invest 101:2497-2505, 1998; McMahon et al., Nat Med 3:3, 2002;Cannon et al., J Clin Invest 108:279-287, 2001; Gladwin et al., J BiolChem 21:21, 2002; Gladwin et al., Circulation 107:271-278, 2003;Schechter et al., N Engl J Med 348:1483-1485, 2003).

In addition to the reaction of nitrite with deoxyhemoglobin, reactionswith deoxy-myoglobin, -cytoglobin and -neuroglobin or with otherendothelial cell heme proteins may also be important. Such chemistrywould occur between tissue nitrite and deoxy-myoglobin in vascular andskeletal muscle, thus contributing to hypoxic vasodilation and hypoxicpotentiation of NO donors. The P₅₀ of these globin monomers isapproximately 3-5 mm Hg, placing their equilibrium deoxygenation pointin the range of tissue pO₂ (0-10 mm Hg) during metabolic stress, such asexercise. Such a low oxygen tension reduces oxygen availability assubstrate for NO synthesis, however, the tissue nitrite stores couldthen be reduced to NO and 5-nitrosothiol, thus sustaining criticalvasodilation.

VII. Methods of Use

Therapeutic application of nitrite now can be used to provide selectivevasodilation in a subject, and particularly to hypoxemic and ischemictissue in the subject, and will be useful to treat hemolytic conditionssuch as sickle cell disease, where free hemoglobin released duringhemolysis scavenges NO and disrupts NO-dependent vascular function.Nitrite is expected to not only inhibit the ability of free hemoglobinto scavenge NO by oxidizing it to methemoglobin, but also to generate NOin tissue beds with low oxygen tension. Thus, the applied nitrite willpreferentially release nitric oxide at areas of low oxygen tension,thereby providing localized vasodilation and/or increased blood flow.

Nitrites can be administered to a subject to increase blood flow to atissue of the subject, for example, to increase blood flow to a tissue,for instance a tissue with low oxygen tension; to cause vasodilation; todecrease a subject's blood pressure; to treat a subject having acondition associated with elevated blood pressure; to treat a hemolyticcondition; to treat vascular complications associated with treatments orconditions that cause hemolysis; to treat pulmonary hypertension,cerebral vasospasm, or low blood flow to organs (such as ischemiareperfusion injury to organs including brain, heart, kidney, placenta,and liver); and/or to treat organs before and after transplantation.

Nitrite has Vasodilatory Properties In Vivo

The vasodilator properties of nitrite and the mechanisms for itsbioactivation were investigated as described herein. Sodium nitriteinfused at 36 μmoles per minute into the forearm brachial artery of 18normal volunteers resulted in a regional nitrite concentration of 222 μMand, surprisingly, a 175% increase in resting forearm blood flow.Increased blood flow was observed at rest, during NO synthase inhibitionand with exercise. The nitrite infusion also surprisingly resulted inincreased tissue perfusion, as demonstrated by increases in venoushemoglobin-oxygen saturation, partial pressure of oxygen, and pH.Increased systemic concentrations of nitrite (16 μM) significantlyreduced mean arterial blood pressure.

In an additional ten subjects, the dose of nitrite was reduced 2-logs,resulting in a forearm nitrite concentration of 2 μM at rest and 0.9 μMduring exercise (FIG. 3). These concentrations of nitrite surprisinglysignificantly increased blood flow at rest and during NO synthaseinhibition, with and without exercise.

Nitrite infusions were associated with the rapid formation oferythrocyte iron-nitrosyl-hemoglobin, and to a lesser extent,S-nitroso-hemoglobin across the forearm vasculature. Formation of theseNO-Hb adducts was inversely proportional to the oxyhemoglobinsaturation. Additionally, vasodilation of rat aortic rings and theformation of both NO gas and NO-modified hemoglobin from the nitritereductase activity of deoxyhemoglobin and deoxygenated erythrocytes wasobserved, a result that links tissue hypoxia, hemoglobin allostery, andnitrite bioactivation. These results indicate that physiological levelsof blood and tissue nitrite are a major bioavailable pool of NO thatcontributes to vaso-regulation and provide a mechanism for hypoxicvasodilation via reaction of vascular nitrite with deoxygenated hemeproteins in tissue and/or the erythrocyte.

The findings described herein that administration of nitrite reducesblood pressure and increases blood flow are unexpected and surprisingbecause published reports to date teach the person of ordinary skill inthe art that pharmacological levels of nitrites (below about 100-200μM), when administered to subjects, lack intrinsic vasodilatoryproperties (Lauer et al., Proc Natl Acad Sci USA, 98:12814-9, 2001).

It is also believed that pharmaceutically acceptable salts of nitritecan be infused into patients with hemolytic disease, such as sickle celldisease, to improve blood flow, limit ischemia-reperfusion tissueinjury, and oxidize cell-free plasma Hb. These effects should be usefulin the treatment of sickle cell vaso-occlusive pain crisis, stroke(brain ischemia) and the acute chest syndrome.

Cytoprotective Effects of Nitrite during Ischemia-Reperfusion of theHeart and Liver

The anion nitrite (NO₂ ⁻) forms as a consequence of nitric oxide (NO)oxidation and is present at concentrations of 0.3-1.0 μM in plasma and1-20 μM in tissue (Gladwin et al., Proc Natl Acad Sci USA97:11482-11487, 2000; Rodriguez et al., Proc Natl Acad Sci USA100:336-341, 2003; Rassaf et al., Nat Med 9:481-483, 2003; Bryan et al.,Proc Natl Acad Sci USA, 2004; Gladwin et al., J Clin Invest 113:19-21,2004). Nitrite has been historically considered an inert metabolic endproduct with limited intrinsic biological activity (Lauer et al., ProcNatl Acad Sci USA 98:12814-12819, 2001; McMahon, N Engl J Med349:402-405; author reply 402-405, 2003; Pawloski, N Engl J Med349:402-405; author reply 402-405, 2003). Recent data from our group andothers suggest that nitrite may be reduced to NO during hypoxia andacidosis (Gladwin et al., Proc Natl Acad Sci USA 97:11482-11487, 2000;Bryan et al., Proc Natl Acad Sci USA, 2004; Cosby et al., Nat Med9:1498-1505, 2003; Nagababu et al., J Biol Chem 278:46349-46356, 2003;Tiravanti et al., J Biol Chem 279:11065-11073, 2004). At extremely lowtissue pH and PO₂, nitrite may be reduced to NO by disproportionation(acidic reduction; Zweier et al., Nat Med 1:804-809, 1995) or by theenzymatic action of xanthine oxidoreductase (Millar et al., FEBS Lett427:225-228, 1998; Zhang et al., Biochem Soc Trans 25:524S, 1997; Godberet al., J Biol Chem 275:7757-7763, 2000; Li et al., J Biol Chem276:24482-24489, 2001).

Nitrite represents a circulating and tissue storage form of nitric oxide(NO) whose bioactivation is mediated by the nitrite reductase activitiesof deoxyhemoglobin. Because the rate of NO generation from nitrite islinearly dependent on reductions in oxygen and pH, we hypothesized thatnitrite would be reduced to NO in ischemic tissue and exert NO-dependentprotective effects. Solutions of sodium nitrite were administered in thesetting of hepatic and cardiac ischemia-reperfusion (I/R) injury inmice. In hepatic I/R, nitrite exerted profound dose dependent protectiveeffects on cellular necrosis and apoptosis with highly significantprotective effects observed at near-physiological nitrite concentrations(0.6 μM). In myocardial I/R injury, nitrite reduced cardiac infarct sizeby 67% and significantly improved post-ischemic left ventricularejection fraction. Consistent with hypoxia dependent nitritebioactivation, nitrite was reduced to NO, S-nitrosothiols,N-nitrosamines and iron-nitrosylated heme proteins within 1-30 minutesof reperfusion. Nitrite-mediated protection was dependent on NOgeneration and independent of eNOS and HO-1. These results suggest thatnitrite is a biological storage reserve of NO subserving a criticalfunction in tissue protection from ischemic injury. These studies evincean unexpected and novel therapy for diseases such as myocardialinfarction, organ preservation and transplantation, and shock states.

Although reperfusion of ischemic tissues provides oxygen and metabolicsubstrates necessary for the recovery and survival of reversibly injuredcells, reperfusion itself actually results in the acceleration ofcellular necrosis (Braunwald et al., J. Clin. Invest. 76:1713-1719,1985). Ischemia-reperfusion is characterized by the formation of oxygenradicals upon reintroduction of molecular oxygen to ischemic tissuesresulting in widespread lipid and protein oxidative modifications ofcellular proteins, mitochondrial injury, and tissue apoptosis andnecrosis (McCord et al., Adv Myocardiol 5:183-189, 1985). In addition,following reperfusion of ischemic tissues blood flow may not returnuniformly to all portions of the ischemic tissues, a phenomenon that hasbeen termed the “no-reflow” phenomenon (Kloner et al., J Clin Invest54:1496-1508, 1974). Reductions in blood flow following reperfusion arethought to contribute to cellular injury and necrosis (Kloner et al., JClin Invest 54:1496-1508, 1974). The sudden re-introduction of bloodinto ischemic tissue also results in a dramatic increase in calciumdelivery to the previously ischemic tissue (i.e., “calcium paradox”)resulting in massive tissue disruption, enzyme release, reductions inhigh energy phosphate stores, mitochondrial injury, and necrosis(Nagler, Amer. J. Path. 102:262, 1981; Shen et al., Amer. J. Path67:417-440, 1972). Recent studies have also indicated that theischemia-reperfusion injury is also characterized by an inappropriateinflammatory response in the microcirculation resulting inleukocyte-endothelial cell interactions that are mediated by theupregulation of both leukocyte and endothelial cell adhesion molecules(Lefer et al., Cardiovasc Res 32:743-751, 1996; Entman et al., Faseb J5:2529-2537, 1991). Intensive research efforts have been focused onameliorating various pathophysiological components ofischemia-reperfusion injury to limit the extent of tissue injury andnecrosis.

NO, NO donors, and NO synthase activation or transgenic over-expressionhave been shown to exert protective effects on this process in a numberof models (Lefer et al., New Horiz 3:105-112, 1995; Lefer et al.,Circulation 88:2337-2350, 1993; Nakanishi et al., Am J Physiol263:H1650-1658, 1992; Jones et al., Am J Physiol Heart Circ Physiol286:H276-282, 2004; Jones et al., Proc Natl Acad Sci USA 100:4891-4896.2003; Kanno et al., Circulation 101:2742-2748, 2000), but in othermodels appears harmful (Flogel et al., J Mol Cell Cardiol 31:827-836.1999; Menezes et al., Am J Physiol 277:G144-151, 1999; Woolfson et al.,Circulation 91:1545-1551, 1995; Schulz, R. et al., Cardiovasc Res30:432-439, 1995). Evaluation of these studies suggests a criticaleffect of dose and duration of NO exposure, resulting in a narrowtherapeutic safety window for NO in ischemia-reperfusion pathophysiology(Bolli, J. Mol. Cell. Cardio. 33:1897-1918, 2001; Wink et al., Am JPhysiol Heart Circ Physiol 285:H2264-2276, 2003). An additionallimitation is that NO formation from NO synthase requires oxygen assubstrate, a molecule whose availability becomes limited duringischemia.

We therefore considered the use of nitrite in this context for thefollowing reasons: (1) It is a naturally occurring substance with nopotentially toxic “leaving group” (2), it is selectively reduced to NOin tissues with low oxygen tension and low pH (Bryan et al., Proc NatlAcad Sci USA, 2004; Cosby et al., Nat Med 9:1498-1505, 2003; Nagababu etal., J Biol Chem 278:46349-46356, 2003; Tiravanti et al., J Biol Chem279:11065-11073, 2004; Doyle et al., J Biol Chem 256:12393-12398, 1981;Luchsinger et al., Proc Natl Acad Sci USA 100:461-466, 2003), (3) itsactivation does not require molecular oxygen (Cosby et al., Nat Med9:1498-1505, 2003), and (4) NO is known to maintain heme proteins in areduced and liganded state (Herold et al., Free Radic Biol Med34:531-545, 2003; Herold et al., J Biol Inorg Chem 6:543-555, 2001;Fernandez et al., Inorg Chem 42:2-4, 2003), limit free iron and hememediated oxidative chemistry (Kanner et al., Arch Biochem Biophys237:314-321, 1985; Kanner et al., Lipids 20:625-628, 1985; Kanner etal., Lipids 27:46-49, 1992), transiently inhibit cytochome c oxidase andmitochondrial respiration (Torres et al., FEBS Lett 475:263-266, 2000;Brown et al., FEBS Lett 356:295-298, 1994; Cleeter et al., FEBS Lett345:50-54, 1994; Rakhit et al., Circulation 103:2617-2623, 2001), andmodulate apoptotic effectors (Mannick et al., Science 284:651-654,1999), all mechanisms that might participate in cytotoxicity followingsevere ischemia.

Nitric oxide has been shown to quench oxygen free radicals in atransient ischemia and reperfusion injury animal models (Mason et al., JNeurosurg 93: 99-107, 2000), significantly limiting volume of stroke(Pluta et al., Neurosurgery, 48:884-892, 2001). Therefore, nitrite viareleasing NO in the area of reperfusion may also have the samebeneficial effect on stroke via limiting oxygen free radicals presenceafter reperfusion.

Furthermore, the selective opening of blood-tumor barrier by NOfacilitates penetration of chemotherapeutic agents into the brain tumor(Weyerbrock et al., J. Neurosurgery, 99:728-737, 2003); it is believedthat this will also enhance penetration of other agents, particularlytherapeutic agents such as radiation therapy, brain cancer. Therefore,due to hypoxic conditions within the brain tumor it is possible thatnitrite can also selectively open the blood-tumor barrier providingbeneficial effect in combination with chemotherapy.

Inhaled Nebulized Nitrite is a Pulmonary Vasodilator

Persistent pulmonary hypertension of the newborn occurs with anincidence of 0.43-6.8/1,000 live births and is associated with mortalityrates between 10-20% (Walsh-Sukys et al., Pediatrics 105, 14-20, 2000).Survivors may develop neurodevelopmental and audiological impairment(46%), cognitive delays (30%), hearing loss (19%) and a high rate ofrehospitalization (22%) (Lipkin et al., J Pediatr 140, 306-10, 2002).

Pulmonary hypertension occurs as a primary or idiopathic disease (Runo &Loyd, Lancet 361:1533-44, 2003; Trembath & Harrison, Pediatr Res53:883-8, 2003), as well as secondary to a number of systemic andpulmonary diseases (Rubin, N Engl J Med 336:111-7, 1997). Regardless ofetiology, pulmonary hypertension is associated with substantialmorbidity and mortality. Newborn infants and adults with pulmonarydisease often develop systemic hypoxemia, reduced oxyhemoglobinsaturation and increased pulmonary vascular resistance (Rubin, N Engl JMed 336:111-7, 1997; Haworth, Heart 88:658-64, 2002). Therapeuticallyadministered inhaled nitric oxide (NO) decreases pulmonary vascularresistance in newborns and adults and improves ventilation-to-perfusionmatching and oxygenation; in newborns, inhaled NO reduces chronic lungdamage and reduces the need for extracorporeal membrane oxygenation.Randomized placebo-controlled trials of inhaled NO therapy for term andnear-term newborns with severe hypoxic respiratory failure demonstratedan improvement in hypoxemia and reduced need for extracorporal membraneoxygenation (Clark et al., N Engl J Med 342, 469-74, 2000; Roberts etal., N Engl J Med 336, 605-10, 1997; The Neonatal Inhaled Nitric OxideStudy Group. N Engl J Med 336, 597-604, 1997). A recent randomizedplacebo-controlled trial in premature infants with respiratory distresssyndrome indicated that treatment with inhaled NO reduced the combinedendpoint of death and chronic lung disease (Schreiber et al., N Engl JMed 349, 2099-107, 2003).

Despite the encouraging results regarding treatment of persistentpulmonary hypertension of the newborn with inhaled NO, the therapy doeshave several significant limitations (Martin, N Engl J Med 349, 2157-9,2003): considerable cost (Jacobs et al., Crit Care Med 30, 2330-4, 2002;Pierce et al.,. Bmj 325, 336, 2002; Subhedar et al., Lancet 359, 1781-2,2002; Angus et al., Pediatrics 112, 1351-60, 2003), technicaldifficulties involved in adapting NO delivery systems for neonataltransport (Kinsella et al., Pediatrics 109, 158-61, 2002), and the lackof availability in small community hospitals and developing countries.In addition, NO reacts with oxygen, forming the toxic nitrogen dioxide,and thus must be stored and delivered in nitrogen at high flow rates.The gas and delivery systems are costly and the requisite deliverytechnology is not universally available. Therefore, alternative NO-basedtherapies for the treatment of pulmonary hypertension are highlydesirable.

The relationship between nitrite and nitric oxide has been appreciatedfor close to a century, with Haldane and later Hoagland recognizing thatiron-nitrosylated myoglobin (NO bound to heme) formed as an end-productduring nitrite-based meat curing (Gladwin, J Clin Invest 113, 19-21,2004). More than fifty years ago, Furchgott and Bhadrakom reported thatnitrite vasodilated aortic ring preparations in vitro (Furchgott &Bhadrakom, J Pharmacol Exp Ther 108, 129-43, 1953); this observation waslater explored by Ignarro's group in experiments evaluating the role ofsoluble guanylyl cyclase in endothelium-dependent vasodilation (Ignarroet al., J Pharmacol Exp Ther 218, 739-49, 1981). However, the highconcentrations of nitrite, typically in the millimolar range, requiredto elicit vasodilation in aortic ring in vitro bioassays precludedconsideration of nitrite as a physiological vasodilator (Lauer et al.,Proc Natl Acad Sci USA 98, 12814-9, 2001; Pawloski, N Engl J Med 349,402-5; author reply 402-5, 2003; McMahon, N Engl J Med 349, 402-5;author reply 402-5, 2003).

Two decades later, in human physiological studies, we observedartery-to-vein differences for nitrite across the human forearm withincreased extraction occurring during NO inhalation and exercise stresswith concomitant NO synthase inhibition (Gladwin et al., Proc Natl AcadSci USA 97, 11482-7, 2000). This finding suggested that nitrite wasbeing metabolized across the forearm with increased consumption duringexercise. Based on these observations along with data from a number ofinvestigators that identified mechanisms for non-enzymatic (nitritedisproportionation) (Zweier et al., Nat Med 1, 804-9, 1995) andenzymatic (xanthine oxidoreductase) (Zweier et al., Nat Med 1, 804-9,1995; Millar et al., FEBS Lett 427, 225-8, 1998; Tiravanti et al., JBiol Chem 279:11065-11073, 2004; Li et al., J Biol Chem,279(17):16939-16946, 2004) reduction of nitrite to NO, we hypothesizedthat nitrite is reduced in vivo to NO in tissues under conditions of lowPo₂ or pH. We found support for this hypothesis in studies of normalhuman volunteers wherein nitrite infusion into the forearm resulted inmarked vasodilation even under basal conditions at near-physiologicalnitrite concentrations (Example 1; Cosby et al., Nat Med 9, 1498-505,2003). The mechanism of this vasodilation was consistent with a reactionof nitrite with deoxygenated hemoglobin to form NO, methemoglobin (Cosbyet al., Nat Med 9, 1498-505, 2003; Nagababu et al., J Biol Chem 278,46349-56, 2003) and other NO adducts.

This nitrite reductase activity of deoxyhemoglobin was extensivelycharacterized by Doyle and colleagues in 1981 (Doyle et al., J Biol Chem256, 12393-8, 1981): nitrite appears to react with deoxyhemoglobin and aproton to form NO and methemoglobin. Such chemistry is ideally suitedfor hypoxic generation of NO from nitrite, as the reaction is enhancedby hemoglobin deoxygenation and acid, providing a graded production ofNO from nitrite linked to physiological changes in oxygen and pH/CO₂.The observation in this current example that inhaled nitrite generatesiron-nitrosyl-hemoglobin, exhaled NO gas, and produces vasodilation inproportion to decreasing levels of oxygenation and pH further indicatesthat nitrite is a bioavailable storage pool of NO and that hemoglobinmay have a physiological function as a nitrite reductase, potentiallycontributing to hypoxic vasodilation (see Example 1). In addition tothese mechanistic considerations, this example supports anothertherapeutic application of nitrite, extending beyond itswell-established role in the treatment of cyanide poisoning.

We show herein (Example 3) that this biochemical reaction can beharnessed for the treatment of neonatal pulmonary hypertension, anNO-deficient state characterized by pulmonary vasoconstriction,right-to-left shunt pathophysiology, ventilation/perfusion inhomogeneityand systemic hypoxemia. We delivered inhaled sodium nitrite by aerosolto newborn lambs with hypoxic and normoxic pulmonary hypertension.Inhaled nitrite elicited a rapid and sustained reduction (˜60%) inhypoxia induced pulmonary hypertension, a magnitude approaching that ofthe effects of 20 ppm NO gas inhalation and which was associated withthe immediate appearance of increasing levels of NO in expiratory gas.Pulmonary vasodilation elicited by aerosolized nitrite wasdeoxyhemoglobin- and pH-dependent and was associated with increasedblood levels of hemoglobin iron-nitrosylation. Significantly, from atherapeutic standpoint, short term delivery of nitrite, dissolved insaline, via nebulization produced selective and sustained pulmonaryvasodilation with no appreciable increase in blood methemoglobin levels.These data support the paradigm that nitrite is a vasodilator acting viaconversion to NO, a process coupled to hemoglobin deoxygenation andprotonation, and further evince a novel, simple and inexpensivepotential therapy for neonatal pulmonary hypertension.

Aerosolized nitrite is an effective vasodilatory in the describednewborn lamb model (Example 3). It can be readily administered bynebulization, and appears to exhibit a wide therapeutic-to-safetymargin, with limited systemic hemodynamic changes and methemoglobinproduction. This presents an attractive therapeutic option to inhaledNO. Nitrite is an ideal “NO producing” agent in that it 1) is anaturally occurring compound in blood, alveolar lining fluid, andtissue, and 2) has no parent-compound leaving group, such as thediazenium diolates, that requires extensive toxicological study prior totranslation to human disease.

Inhaled nitrite is a potent and selective vasodilator of pulmonarycirculation of the newborn lamb. This further supports the paradigm thatnitrite is an NO-dependent vasodilator whose bioactivation is coupled tohemoglobin deoxygenation and protonation. This has clinical applicationsin veterinary and medical situations, including pulmonary hypertensionand other pulmonary syndromes with apparent NO deficiencies. Based onthe data presented herein, it is believed that inhaled nitrite will haveefficacy in all known and tested applications of inhaled NO.

Prevention of Cerebral Artery Vasospasm after Subarachnoid Hemorrhage

Further, it has been discovered that nitrite infusion can be used toprevent cerebral artery vasospasm after aneurismal hemorrhage (Example4). Subarachnoid hemorrhage (SAH) due to the rupture of intracranialaneurysms affects 28,000 Americans annually. Almost 70% of patients withaneurysmal SAH develop severe spasm of the cerebral arteries on theseventh day after SAH. Despite aggressive medical therapy, neurologicaldeficits resulting from vasospasm continue to be a major cause ofmorbidity and mortality. Although the etiology of cerebral vasospasm ispoorly understood, there is increasing evidence that erythrocytehemolysis in the cerebrospinal fluid and decreased availability ofnitric oxide (NO), a potent vasodilator, plays a significant role.Reversal of vasospasm by NO or NO prodrugs has been documented inseveral animal models.

Delayed cerebral vasospasm (DCV) remains the single cause of permanentneurological deficits or death in at least fifteen percent of patientsfollowing otherwise successful endovascular or surgical treatment forruptured intracranial aneurysm. Decreased bioavailability of nitricoxide (NO) has been mechanistically associated with the development ofDCV. A primate model system for cerebral artery vasospasm was used todetermine whether infusions of nitrite, a naturally occurring anion thatreacts with deoxyhemoglobin to form NO and S-nitrosothiol, might preventDCV via reactions with perivascular hemoglobin.

As described in Example 4, nitrite infusions (45 mg/kg and 60 mg/kg perday) that produced blood levels of nitrite ranging from 10-60 microMwith no clinically significant methemoglobin formation (<5%) wereassociated with increases in plasma cerebrospinal fluid nitrite andmodest increases in blood methemoglobin concentrations (2% or less)without systemic hypotension, and significantly reduced the severity ofvasospasm (FIGS. 15 and 16). No animals infused with sodium nitritedeveloped significant vasospasm; mean reduction in the R MCA area on day7 after SAH was 8±9% versus 45±5%; P<0.001) Pharmacological effects ofnitrite infusion were associated with bioconversion of cerebrospinalfluid nitrite to S-nitrosothiol, a potent vasodilating NO donorintermediate of nitrite bioactivation. There was no clinical orpathological evidence of nitrite toxicity.

Subacute sodium nitrite infusions prevent DCV in a primate model of SAH,and do so without toxicity. These data evince a novel, safe,inexpensive, and rationally designed therapy for DCV, a disease forwhich no current preventative therapy exists.

The results presented herein suggest that sodium nitrite therapy mayprevent tissue injury produced by metabolic products of hemoglobin,either by vascular spasm, or by other mechanisms of tissue injury bythese metabolic products.

Treatment or Amelioration of Gestational or Fetal CardiovascularMalconditions

Based on results presented herein, it is believed that nitrite,particularly pharmaceutically acceptable salts of nitrite as describedherein, can be used to treat hypertension and preeclampsia duringpregnancy. Such therapy would include action of nitrites on spastic anddiseased blood vessels within the placenta.

Also suggested are methods for treating fetuses in utero, particularlythose afflicted with cardiovascular anomalies, hypertension, andmisdirected blood flow. It is believed that it may be possible to addnitrites to the amniotic fluid, and thus indirectly to the fetus, toachieve vasodilation and redistribution of blood flow before birth. Bythis means, fetal cardiovascular system development and function couldbe altered, for instance with promotion of blood flow to the brain andheart. To be effective longer term, it is envisioned that embodiments ofsuch fetal therapy would include the introduction of one or moremini-osmotic pumps, containing nitrite (e.g., sodium nitrite), into theamniotic cavity to thereby achieve sustained, slow release. Forinstance, such minipumps could be used to achieve sustained releasethroughout days and weeks of pregnancy.

Also suggested are methods for treating fetuses in whom plasma nitritelevels may be depressed by immune incompatibility and associatedhemolytic anemias. Such fetal treatment may be extended into theneonatal period. Administrated in the fetal period may includeimplantation of nitrite-charged osmotic minipumps into the amnioticcavity and could include aerosol inhalation after birth.

VIII. Formulations and Administration

Nitrites, including their salts, are administered to a subject inaccordance to methods provided herein, in order to decrease bloodpressure and/or increase vasodilation in a subject. Administration ofthe nitrites in accordance with the present disclosure may be in asingle dose, in multiple doses, and/or in a continuous or intermittentmanner, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of the nitrites may be essentially continuous over apreselected period of time or may be in a series of spaced doses. Theamount administered will vary depending on various factors including,but not limited to, the condition to be treated and the weight, physicalcondition, health, and age of the subject. Such factors can bedetermined by a clinician employing animal models or other test systemsthat are available in the art.

To prepare the nitrites, nitrites are synthesized or otherwise obtainedand purified as necessary or desired. In some embodiments of thedisclosure, the nitrite is a pharmaceutically-acceptable salt ofnitrite, for example, sodium nitrite. In some embodiments of thedisclosure, the nitrite is not ethyl nitrite. In some embodiments of thedisclosure, the sodium nitrite is not on a medical devise, for example,not on a stent. In some embodiments of the disclosure, the nitrite isnot in the form of a gel. The nitrites can be adjusted to theappropriate concentration, and optionally combined with other agents.The absolute weight of a given nitrite included in a unit dose can vary.In some embodiments of the disclosure, the nitrite is administered as asalt of an anionic nitrite with a cation, for example, sodium,potassium, or arginine.

One or more suitable unit dosage forms including the nitrite can beadministered by a variety of routes including topical, oral (forinstance, in an enterically coated formulation), parenteral (includingsubcutaneous, intravenous, intramuscular and intraperitoneal), rectal,intraamnitic, dermal, transdermal, intrathoracic, intrapulmonary andintranasal (respiratory) routes.

The formulations may, where appropriate, be conveniently presented indiscrete unit dosage forms and may be prepared by any of the methodsknown to the pharmaceutical arts. Such methods include the step ofmixing the nitrite with liquid carriers, solid matrices, semi-solidcarriers, finely divided solid carriers or combinations thereof, andthen, if necessary, introducing or shaping the product into the desireddelivery system. By “pharmaceutically acceptable” it is meant a carrier,diluent, excipient, and/or salt that is compatible with the otheringredients of the formulation, and not deleterious or unsuitablyharmful to the recipient thereof. The therapeutic compounds may also beformulated for sustained release, for example, using microencapsulation(see WO 94/07529, and U.S. Pat. No. 4,962,091).

The nitrites may be formulated for parenteral administration (e.g., byinjection, for example, bolus injection or continuous infusion) and maybe presented in unit dose form in ampoules, pre-filled syringes, smallvolume infusion containers or in multi-dose containers. Preservativescan be added to help maintain the shelve life of the dosage form. Thenitrites and other ingredients may form suspensions, solutions, oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the nitrites and other ingredients may be in powder form,obtained by aseptic isolation of sterile solid or by lyophilization fromsolution, for constitution with a suitable vehicle, e.g., sterile,pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable carriers andvehicles that are available in the art. It is possible, for example, toprepare solutions using one or more organic solvent(s) that is/areacceptable from the physiological standpoint, chosen, in addition towater, from solvents such as acetone, ethanol, isopropyl alcohol, glycolethers such as the products sold under the name “Dowanol,” polyglycolsand polyethylene glycols, C₁-C₄ alkyl esters of short-chain acids, ethylor isopropyl lactate, fatty acid triglycerides such as the productsmarketed under the name “Miglyol,” isopropyl myristate, animal, mineraland vegetable oils and polysiloxanes.

It is possible to add other ingredients such as antioxidants,surfactants, preservatives, film-forming, keratolytic or comedolyticagents, perfumes, flavorings and colorings. Antioxidants such ast-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytolueneand α-tocopherol and its derivatives can be added.

The pharmaceutical formulations of the present disclosure may include,as optional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that areavailable in the art. Examples of such substances include normal salinesolutions such as physiologically buffered saline solutions and water.Specific non-limiting examples of the carriers and/or diluents that areuseful in the pharmaceutical formulations of the present disclosureinclude water and physiologically acceptable buffered saline solutions,such as phosphate buffered saline solutions. Merely by way of example,the buffered solution can be at a pH of about 6.0-8.5, for instanceabout 6.5-8.5, about 7-8.

The nitrites can also be administered via the respiratory tract. Thus,the present disclosure also provides aerosol pharmaceutical formulationsand dosage forms for use in the methods of the disclosure. In general,such dosage forms include an amount of nitrite effective to treat orprevent the clinical symptoms of a specific condition. Any attenuation,for example a statistically significant attenuation, of one or moresymptoms of a condition that has been treated pursuant to the methods ofthe present disclosure is considered to be a treatment of such conditionand is within the scope of the disclosure.

For administration by inhalation, the composition may take the form of adry powder, for example, a powder mix of the nitrite and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form in, for example, capsules or cartridges,or, e.g., gelatin or blister packs from which the powder may beadministered with the aid of an inhalator, insufflator, or ametered-dose inhaler (see, for example, the pressurized metered doseinhaler (MDI) and the dry powder inhaler disclosed in Newman, S. P. inAerosols and the Lung, Clarke, S. W. and Davia, D. eds., pp. 197-224,Butterworths, London, England, 1984).

Nitrites may also be administered in an aqueous solution, for example,when administered in an aerosol or inhaled form. Thus, other aerosolpharmaceutical formulations may include, for example, a physiologicallyacceptable buffered saline solution. Dry aerosol in the form of finelydivided solid compound that is not dissolved or suspended in a liquid isalso useful in the practice of the present disclosure.

For administration to the respiratory tract, for example, the upper(nasal) or lower respiratory tract, by inhalation, the nitrites can beconveniently delivered from a nebulizer or a pressurized pack or otherconvenient means of delivering an aerosol spray. Pressurized packs mayinclude a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Nebulizers include, but are not limited to, those described in U.S. Pat.Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol deliverysystems of the type disclosed herein are available from numerouscommercial sources including Fisons Corporation (Bedford, Mass.),Schering Corp. (Kenilworth, N.J.) and American Pharmoseal Co. (Valencia,Calif.). For intra-nasal administration, the therapeutic agent may alsobe administered via nose drops, a liquid spray, such as via a plasticbottle atomizer or metered-dose inhaler. Typical of atomizers are theMistometer (Wintrop) and the Medihaler (Riker). The nitrites may also bedelivered via an ultrasonic delivery system. In some embodiments of thedisclosure, the nitrites may be delivered via an endotracheal tube. Insome embodiments of the disclosure, the nitrites may be delivered via aface mask.

The present disclosure further pertains to a packaged pharmaceuticalcomposition such as a kit or other container. The kit or container holdsa therapeutically effective amount of a pharmaceutical composition ofnitrite and instructions for using the pharmaceutical composition fortreating a condition.

IX. Combination Therapies

Furthermore, the nitrite may also be used in combination with othertherapeutic agents, for example, pain relievers, anti-inflammatoryagents, antihistamines, and the like, whether for the conditionsdescribed or some other condition. By way of example, the additionalagent is one or more selected from the list consisting of penicillin,hydroxyurea, butyrate, clotrimazole, arginine, or a phosphodiesteraseinhibitor (such as sildenafil).

Generally, it is believed that therapies that have been suggested ordemonstrated to be effective when combined with NO therapy, may also beeffective when combined with nitrite administration. All combinationtherapies that have been are being studied with NO therapy (inhaled orotherwise) are likely to be worthy of study in combination with nitritetherapy. See, for instance, Uga et al., Pediatr. Int. 46 (1): 10-14,2004; Gianetti et al., J Thorac. Cardiov. Sur. 127 (1): 44-50, 2004;Stubbe et al., Intens. Care Med. 29 (10): 1790-1797, 2003; Wagner etal., Eur. Heart J 23: 326-326 Suppl. 2002; Park et al., Yonesi Med J 44(2):219-226, 2003; Kohele, Israel Med. Assoc. J. 5:19-23, 2003, fordiscussions of combination therapies used with NO.

Furthermore, pharmaceutically-acceptable nitrite salts (such as, forinstance, sodium nitrite) may be used in combinations with drugs andagents that limit the elimination rate of administered nitrites. Thiscombination could serve to prolong the duration of action of nitrite andwould include antagonists and inhibitors of enzymes affecting theelimination of nitrites or their conversion to NO.

Alternatively, the nitrite may be used in combinations with drugs andagents that augment the action of nitrites. This combination could serveto increase the strength of responses to administered nitrites.

Recombinant tissue plasminogen activator (rt-PA) and urokinase are theonly drugs that have proven to open occluded brain arteries in ischemicstroke. It is believed possible that using nitrite via quenching oxygenfree radicals produced in response to reperfusion may provide anadditional beneficial effect.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

Example 1 Nitrite has Vasodilatory Properties In Vivo

This example provides a demonstration that nitrite, administered byinfusion to the forearm of human subjects, is an effective vasodilator.

Methods

Human Subjects Protocol.

The protocol was approved by the Institutional Review Board of theNational Heart, Lung and Blood Institute, and informed consent wasobtained from all volunteer subjects. Nine men and nine women, with anaverage age of 33 years (range 21-50 years), participated in the study.An additional 10 subjects returned three-six months later for a secondseries of experiments with low dose nitrite infusion. Volunteers had anormal hemoglobin concentration, and all were in excellent generalhealth without risk factors for endothelial dysfunction (fasting bloodsugar>120 mg/dL, low-density lipoprotein cholesterol>130 mg/dL, bloodpressure>145/95 mmHg, smoking within two years, cardiovascular disease,peripheral vascular disease, coagulopathy, or any other diseasepredisposing to vasculitis or Raynaud's phenomenon). Subjects with G6PDdeficiency, known cytochrome B5 deficiency or a baseline methemoglobinlevel>1% were excluded (no screened subjects met these exclusioncriteria). Lactating and pregnant females were excluded (one subjectwith positive HCG levels was excluded). No volunteer subject was allowedto take any medication (oral contraceptive agents allowed), vitaminsupplements, herbal preparations, nutraceuticals or other “alternativetherapies” for at least one month prior to study and were not be allowedto take aspirin for one week prior to study.

Forearm Blood Flow Measurements

Brachial artery and antecubital vein catheters were placed into the arm,with the intra-arterial catheter connected to a pressure transducer forblood pressure measurements and an infusion pump delivering normalsaline at 1 mL/min. After 20 minutes of rest, baseline arterial andvenous blood samples were obtained and forearm blood flow measurementswere made by strain gauge venous-occlusion plethysmography, aspreviously reported (Panza et al., Circulation, 87, 1468-74, 1993). Aseries of 7 blood flow measurements were averaged for each blood flowdetermination. A series of measurements termed Parts I and II wereperformed in randomized order to minimize a time effect on the forearmblood flow response during nitrite infusion.

Measurement of Blood Flow and Forearm Nitrite Extraction During NOBlockade and Repetitive Exercise

Part I: Following 20 minutes of 0.9% NaCl (saline) solution infusion at1 mL/min into the brachial artery, arterial and venous blood sampleswere obtained for the assays described below and forearm blood flowmeasured. Exercise was performed by repetitive hand-grip at one-third ofthe predetermined maximum grip strength using a hand-grip dynamometer(Technical Products Co.) (Gladwin et al., Proc Natl Acad Sci USA, 97,9943-8, 2000; Gladwin et al., Proc Natl Acad Sci USA, 97, 11482-11487,2000; Cannon et al., J Clin Invest, 108, 279-87, 2001). Each contractionlasted for 10 seconds followed by relaxation for 5 seconds. Following 5minutes of exercise, forearm blood flow measurements were obtainedduring relaxation phases of exercise, and arterial and venous samplescollected. Following a 20-minute rest period with continued infusion ofsaline into the brachial artery, repeated baseline blood samples andforearm blood flow measurements were obtained. L-NMMA was then infusedat a rate of 1 mL/min (8 μmol/min) into the brachial artery. Following 5minutes of L-NMMA infusion, forearm blood flow was measured, andarterial and venous blood samples obtained. Forearm exercise was theninitiated in that arm during continued L-NMMA infusion. Forearm bloodflow was measured and blood samples obtained after 5 minutes of exerciseduring continued L-NMMA infusion (FIG. 1).

Part II: After a 30 minute rest period with continued infusion ofsaline, baseline measurements were obtained, the saline infusion wasthen stopped, and infusion of nitrite (NaNO₂ 36 μmol/ml in 0.9% saline)at 1 ml/min was started. Sodium nitrite for use in humans was obtainedfrom Hope Pharmaceuticals (300 mg in 10 ml water) and 286 mg was dilutedin 100 ml 0.9% saline by the Pharmaceutical Development Service to afinal concentration of 36 μmol/ml. For the final 9 subjects studied,0.01-0.03 mM sodium bicarbonate was added to the normal saline, so as totitrate pH to 7.0-7.4. The nitrite solution was light protected andnitrite levels and free NO gas in solution measured by reductivechemiluminescence after all experiments (Gladwin et al., J Biol Chem,21, 21, 2002). Only 50.5±40.5 nM NO was present in nitrite solutions andwas unaffected by bicarbonate buffering. There was no correlationbetween NO levels in nitrite solutions and blood flow effects of nitrite(r=−0.23; P=0.55). After 5 minutes of nitrite infusion, forearm bloodflow measurements and blood samples were obtained, with briefinterruption of the nitrite infusion to obtain the arterial sample. Withcontinued nitrite infusion, exercise was performed as describedpreviously, with forearm blood flow measurements and blood samplesobtained as described above. The nitrite infusion was stopped and salineinfusion re-started during the subsequent 30-minute rest period.Following second baseline measurements, the nitrite infusion wasre-initiated, along with L-NMMA at 8 μmol/min. Five minutes later,forearm blood flow measurements were performed and blood samplesobtained followed by 5 minutes of exercise with continuation of nitriteand L-NMMA infusions. Final forearm blood flow measurements and bloodsamples obtained. At all time points during part II, blood samples wereobtained from the contralateral arm antecubital vein for determinationof methemoglobin and systemic levels of NO-modified hemoglobin (FIGS. 2,3, and 4). The total dose of sodium nitrite infused was 36μmol/minute×15 minutes×2 infusions =1.08 mmol=75 mg (MW NaNO₂=69).

In additional studies in 10 subjects the same stages of Parts I and IIprotocol were followed with infusion of low dose nitrite (NaNO₂ 0.36mol/ml in 0.9% saline, infused at 1 ml/min).

Arterial and venous pH, pO₂, and pCO₂, were measured at the bedsideusing the i-STAT system (i-STAT Corporation, East Windsor, N.J.) andmethemoglobin concentration and hemoglobin oxygen saturation measured byco-oximetry.

Measurement of Red Blood Cell S-nitroso-hemoglobin andIron-nitrosyl-hemoglobin.

S-nitroso-hemoglobin is unstable in the reductive red blood cellenvironment and rapidly decays in a temperature and redox dependentfashion, independent of oxygen tension (Gladwin et al., J Biol Chem,21:21, 2002). To stabilize the S-nitroso-hemoglobin for measurement, thered blood cell must be rapidly oxidized with ferricyanide. Before andduring nitrite infusions, blood was drawn from both the brachial arteryand antecubital vein and the whole blood immediately (at the bedside toeliminate processing time) lysed 1:10 in an NO-hemoglobin “stabilizationsolution” of PBS containing 1% NP-40 (to solubilize membranes), 8 mM NEM(to bind free thiol and prevent artefactual S-nitrosation), 0.1 mM DTPA(to chelate trace copper), and 4 mM ferricyanide and cyanide (tostabilize S-nitrosohemoglobin and prevent artefactual ex-vivoiron-nitrosylation during processing). The samples were desalted acrossa 9.5 mL bed volume Sephadex G25 column to eliminate nitrite and excessreagents and partially purify hemoglobin (99% hemoglobin preparation).The hemoglobin fraction was quantified by the method of Drabkin, andhemoglobin fractions reacted with and without mercuric chloride (1:5HgCl₂:heme ratio-used to differentiate S-nitrosothiol which is mercurylabile versus iron-nitrosyl which is mercury stable) and then in 0.1 MHCL/0.5% sulfanilamide (to eliminate residual nitrite; Marley et al.,Free Radic Res, 32, 1-9, 2000). The samples were then injected into asolution of tri-iodide (I₃ ⁻) in-line with a chemiluminescent nitricoxide analyzer (Sievers, Model 280 NO analyzer, Boulder, Colo.). Themercury stable peak represents iron-nitrosyl-hemoglobin. This assay issensitive and specific for both S-nitroso-hemoglobin andiron-nitrosyl-hemoglobin to 5 nM in whole blood (0.00005% S-NO per heme)(Gladwin et al., J Biol Chem, 21, 21, 2002).

Analysis was initially performed using red blood cell pellet, however,despite placing the sample in ice and immediately separating plasma fromerythrocyte pellet, NO formed in the venous blood ex vivo. To measurethe true in vivo levels, whole blood was mixed at the bedside 1:10 inthe “NO-hemoglobin stabilization solution”. Plasma S-nitroso-albuminformation was negligible during nitrite infusion so this bedside wholeblood assay was used to limit processing time and thus more accuratelycharacterize the in vivo chemistry. In a series of validationexperiments, both S-nitroso-hemoglobin and iron-nitrosyl-hemoglobin werestable in the “NO-hemoglobin stabilization solution” for 20 minutes atroom temperature with no artifactual formation or decay of NO-modifiedspecies (n=6).

Chemiluminescent Detection of NO Gas Released from Deoxyhemoglobin andDeoxygenated Erythrocytes Following Nitrite Addition.

To determine whether free NO radical can form from the reaction ofnitrite and deoxyhemoglobin, 100 and 200 μM nitrite was mixed with 5 mLof 660 and 1000 μM deoxygenated erythrocytes in a light protectedreaction vessel purged with helium or oxygen (both 21% and 100%) in-linewith a chemiluminescent NO analyzer (Seivers, Boulder, Colo.). Afterallowing equilibration for 5 minutes, nitrite was injected and the rateof NO production measured. Nitrite was injected into PBS as a controland into 100 μM hemoglobin to control for the hemolysis in the 660 and1000 μM deoxygenated erythrocyte solutions. At the end of allexperiments the visible absorption spectra of the supernatant anderythrocyte reaction mixture was analyzed and hemoglobin compositiondeconvoluted using a least-squares algorithm. There was less than 100 μMhemolysis in the system, no hemoglobin denaturation, and significantformation of iron-nitrosyl-hemoglobin. The NO production fromerythrocyte suspensions exceeded that produced from the hemolysatecontrol, consistent with NO export from the erythrocyte.

Statistical Analysis.

An a priori sample size calculation determined that 18 subjects would benecessary for the study to detect a 25% improvement in forearm bloodflow during nitrite infusion when forearm NO synthesis had beeninhibited by L-NMMA compared with normal saline infusion control values(alpha=0.05, power=0.80). Two-sided P values were calculated by pairedt-test for the pair-wise comparisons between baseline and L-NMMAinfusion values, between baseline and exercise values, and betweennitrite and saline control values at comparable time-points of thestudy. Repeated measures ANOVA were performed for artery-to-veingradients of NO species during basal, L-NMMA infusion, and exerciseconditions. Measurements shown are mean±SEM.

Results and Discussion

Eighteen healthy subjects (9 males, 9 females; age range 21 to 50 years)were enrolled in a physiological study to determine if nitrite is avasodilator and to examine nitrite's in vivo chemistry. Part I of theprotocol was designed to measure the normal hemodynamic and metabolicresponses to exercise and to inhibition of NO synthesis within theforearm as a control for Part II of the protocol, in which theseinterventions were performed during nitrite infusion. Initial baselinemeasurements included a mean blood pressure of 85.6±3.7 mm Hg andforearm blood flow of 4.0±0.3 ml/min per 100 mL tissue (FIG. 1A).Repetitive hand-grip forearm exercise increased blood flow approximately600% over resting values, and significantly decreased ipsilateral venoushemoglobin oxygen saturation, p0₂, and pH, consistent with increasedoxygen consumption and CO₂ generation. Following a 20-minute restperiod, repeat hemodynamic measurements showed an approximate 10% higherforearm blood flow, but no change in systemic blood pressure or forearmvenous hemoglobin oxygen saturation, p0₂ and pH values compared with theinitial baseline values (FIG. 1B). The NO synthase inhibitor L-NMMA wasthen infused into the brachial artery at 8 μmol/min for 5 minutes,significantly reducing forearm blood flow by approximately 30% andsignificantly reducing venous hemoglobin oxygen saturation, p0₂ and pHvalues. Repeated forearm exercise during continued L-NMMA infusionincreased blood flow, but to a significantly lower peak value comparedwith exercise alone (P<0.001). In addition, hemoglobin oxygensaturation, p0₂ and pH were significantly lower during exercise withL-NMMA than with exercise without regional NO synthase inhibition(P<0.001, P<0.005 and P=0.027, respectively). Mean arterial bloodpressure was unchanged during all components of Part I of the protocol.

FIG. 1 depicts hemodynamic and metabolic measurements at baseline andduring exercise, without (FIG. 1A) and with (FIG. 1B) inhibition of NOsynthesis in 18 subjects. Mean arterial pressure (MAP), forearm bloodflow (FBF), and venous oxyhemoglobin saturation, partial pressure ofoxygen (pO₂), and pH are shown for all experimental conditions. Theseinterventions and measurements (part I of the protocol) served as acontrol for Part II of the protocol, in which these interventions wereperformed during nitrite infusion.

To determine whether nitrite has vasoactivity in humans, in Part II ofthe protocol sodium nitrite in bicarbonate-buffered normal saline (finalconcentration 36 μmol/ml) was infused into the brachial arteries ofthese 18 subjects to achieve an estimated intravascular concentration ofapproximately 200 μM (Lauer et al., Proc Natl Acad Sci USA, 98, 12814-9,2001). Following repeat baseline measurements and infusion of sodiumnitrite at 1 mL/min for 5 minutes, nitrite levels in the ipsilateralantecubital vein increased from 3.32±0.32 to 221.82±57.59 μM (FIG. 2A).Forearm blood flow increased 175% over resting values; venous hemoglobinoxygen saturation, p0₂ and pH levels significantly increased overpre-infusion values, consistent with increased perfusion of the forearm.

Systemic levels of nitrite were 16 μM as measured in the contralateralarm and were associated with a systemic effect of decreased mean bloodpressure of approximately 7 mm Hg. Consistent with immediate NOgeneration from nitrite during an arterial-to-venous transit,iron-nitrosylated-hemoglobin in the ipsilateral antecubital veinincreased from 55.7±11.4 to 693.4±216.9 nM during the nitrite infusion.During forearm exercise with continuation of the nitrite infusion, bloodflow increased further, with evidence of metabolic stress by virtue ofreduction in forearm venous hemoglobin oxygen saturation, p0₂ and pHlevels from baseline values. Venous nitrite levels declined, consistentwith increased blood flow to the forearm diluting the concentration ofinfused nitrite. Despite decreasing forearm nitrite concentrationsduring exercise, iron-nitrosyl-hemoglobin levels increased (FIG. 2A).

Following cessation of nitrite infusion and substitution of saline asthe intra-arterial infusate for 30 minutes, repeat baseline measurementsshowed persistent elevations in systemic levels of nitrite,iron-nitrosyl-hemoglobin and methemoglobin (FIG. 2B) over valuesobtained prior to the infusion of nitrite almost one hour before. Inaddition, persistence of a vasodilator effect was also apparent, asforearm blood flow was significantly higher (4.79±0.37 versus 3.94±0.38mL/min per 100 mL tissue, P=0.003) and systemic blood pressuresignificantly lower (82.1±3.7 versus 89.2±3.5 mm Hg, P=0.002) thaninitial pre-nitrite infusion values. During re-infusion into thebrachial artery of sodium nitrite 36 mol/ml, combined with L-NMMA 8μmol/min in order to again inhibit regional synthesis of NO, similarvasodilator effects of nitrite on resting and exercise forearm bloodflow were seen as during nitrite infusion without L-NMMA (FIG. 2B). Thisstands in contrast to the vasoconstrictor effect of NO synthaseinhibition with L-NMMA observed in Part I of the protocol (FIG. 1B).Venous nitrite and iron-nitrosyl-hemoglobin levels followed similarpatterns during NO inhibition as during the initial nitrite infusion.

FIG. 2 depicts the effects of infusion of sodium nitrite (NaNO₂) inbicarbonate-buffered normal saline (0.9%; final concentration 36μmol/ml) into the brachial arteries of 18 healthy subjects at 1 ml/minfor 5 minutes at baseline and continued during exercise. FIG. 2A depictsthe effects without inhibition of NO synthesis. FIG. 2B depicts theeffects with inhibition of NO synthesis. Values for mean arterial bloodpressure (MAP), forearm blood flow (FBF), venous oxyhemoglobinsaturation, partial pressure of oxygen (pO₂) and pH, venous nitrite,venous iron-nitrosyl-hemoglobin and venous methemoglobin are shown forall experimental interventions.

As a test of the physiological relevance of vascular nitrite as avasodilator, nitrite concentrations were decreased by 2-logs to 400nmol/mL. An infusion of 1 mL/min for five minutes in 10 subjectssignificantly increased forearm blood flow in all ten subjects from3.49±0.24 to 4.51±0.33 ml/min per 100 mL tissue (FIG. 3A; P=0.0006).Blood flow significantly increased at rest and during NO synthaseinhibition with and without exercise (FIG. 3B; P<0.05 during allconditions). Mean venous nitrite levels increased from 176±17 nM to2564±462 nM following a five-minute infusion and exercise venous nitritelevels decreased to 909±113 nM (secondary to dilutional effects ofincreased flow during exercise; FIG. 3C). Again, the vasodilator effectsof nitrite were paralleled with an observed formation of bothiron-nitrosyl-hemoglobin and S-nitroso-hemoglobin across the forearmcirculation (FIG. 3D; described below). These data indicate that basallevels of nitrite, from 150-1000 nM in plasma to 10,000 nM in vasculartissue, contribute to resting vascular tone and hypoxic vasodilation.

FIG. 3 depicts the effects of infusion of low-dose sodium nitrite inbicarbonate-buffered normal saline into the brachial arteries of 10healthy subjects at baseline and during exercise, without and withinhibition of NO synthesis. FIG. 3A depicts forearm blood flow atbaseline and following a five-minute in fusion of NaNO₂ (0.36 μmol/ml in0.9% saline, infused at 1 ml/min). FIG. 3B depicts forearm blood flowwith and without low-dose nitrite infusion at baseline and during L-NMMAinfusion with and without exercise stress. FIG. 3C depicts venous levelsof nitrite from the forearm circulation at the time of blood flowmeasurements. FIG. 3D depicts venous levels of S-nitroso-hemoglobin(S-NO) and iron-nitrosyl-hemoglobin (Hb-NO) at baseline and followingnitrite infusion during exercise stress.

The vasodilatory property of nitrite during basal blood flow conditions,when tissue pO₂ and pH are not exceedingly low, was unexpected. Theseresults indicate that the previously hypothesized mechanisms for nitritereduction, nitrite disproportionation and xanthine oxidoreductaseactivity, both of which require extremely low pO₂ and pH values nottypically encountered in normal physiology, are complemented in vivo byadditional factors that serve to catalyze nitrite reduction. Whileascorbic acid and other reductants, present in abundance in blood, canprovide necessary electrons for nitrous acid reduction, such that thereaction might occur at physiologically attainable pH levels, it isherein reported that deoxyhemoglobin effectively reduces nitrite to NO,within one half-circulatory time. This mechanism provides a gradedproduction of NO along the physiological oxygen gradient, tightlyregulated by hemoglobin oxygen desaturation.

Intravascular Formation of NO and S-nitrosothiol by Reaction of Nitritewith Intraerythrocytic Deoxyhemoglobin

Before and during nitrite infusions, blood was drawn from both thebrachial artery and antecubital vein and the whole blood immediately (atthe bedside to eliminate processing time) lysed 1:10 in an NO-hemoglobin“stabilization solution” and the iron-nitrosyl-hemoglobin andS-nitroso-hemoglobin content determined by tri-iodide-based reductivechemiluminescence and electron paramagnetic resonance spectroscopy asdescribed in Methods. The baseline levels of S-nitroso-hemoglobin andiron-nitrosyl-hemoglobin were at the limits of detection (<50 nM or0.0005% NO per heme) with no artery-to-vein gradients. Following nitriteinfusion in Part II of the protocol venous levels of bothiron-nitrosyl-hemoglobin and S-nitroso-hemoglobin rose strikingly (FIG.4A). The formation of both NO-hemoglobin adducts occurred across thevascular bed, a half-circulatory time of less than 10 seconds. The rateof NO formation, measured as iron-nitrosyl and S-nitroso-hemoglobin andquantified by subtraction of the arterial from the venous levels withthe difference being multiplied by blood flow, increased greatly duringexercise, despite a significant decrease in the venous concentration ofnitrite secondary to increasing blood flow diluting the regional nitriteconcentration (FIG. 4A; P=0.006 for iron-nitrosyl-hemoglobin and P=0.02for S-nitroso-hemoglobin by repeated measures ANOVA).

FIG. 4A depicts formation of iron-nitrosyl-hemoglobin (black squares)and S-nitroso-hemoglobin (red circles) during nitrite infusion atbaseline, during nitrite infusion and during nitrite infusion withexercise, quantified by subtraction of the arterial from the venouslevels and multiplying the result by blood flow. The formation of bothNO-hemoglobin adducts was inversely correlated with hemoglobin-oxygensaturation in the human circulation during nitrite infusion (foriron-nitrosyl-hemoglobin r=−0.7, p<0.0001, for S-nitroso-hemoglobinr=−0.45, p=0.04) (FIG. 4B). Hemoglobin oxygen saturation was measuredfrom the antecubital vein by co-oximetry. Asterix in all figures signifyP<0.05 by paired t test or repeated measures analysis of variance.

To determine whether free NO radical can form from the reaction ofnitrite and deoxyhemoglobin, 100 and 200 μM nitrite was reacted withdeoxygenated erythrocytes (5 mL volume containing a total of 660 and1000 μM in heme) in a light protected reaction vessel purged with heliumin-line with a chemiluminescent NO analyzer (Seivers, Boulder, Colo.).As shown in FIGS. 5A and 5B, the injection of nitrite into a solution ofdeoxygenated erythrocytes resulted in the liberation of NO into the gasphase. There was no release from nitrite in buffer control under thesame conditions, and significantly less NO was released upon nitriteaddition to oxygenated erythrocytes (21% and 100% oxygen). The observedrate (determined by the assessment of the area under the curve ofincreased steady-state NO generation following nitrite injectioncalculated over 120 seconds) of NO production in the 5 mL reactionvolume was consistent with 47 pM NO production per second (correspondingto an estimated 300 to 500 pM NO production per second in whole blood).While NO formation rates in this experimental system may not beextrapolated to rates of NO formation in vivo, the experiments areconsistent with two important concepts: 1) A fraction of free NO canescape auto-capture by the remaining heme groups; this is likely onlypossible because nitrite is only converted to NO by reaction withdeoxyhemoglobin and its “leaving group” is the met(ferric)heme proteinwhich will limit scavenging and inactivation of NO (Doyle et al., J BiolChem, 256, 12393-12398, 1981); and 2) The rate of NO production isincreased under anaerobic conditions, consistent with anitrite-deoxyhemoglobin reaction.

Example 2 Cytoprotective Effects of Nitrite during Ischemia-reperfusionof the Heart and Liver

As demonstrated in Example 1, nitrite is reduced to NO by reaction withdeoxyhemoglobin along the physiological oxygen gradient, a chemistrywhose rate is oxygen and pH dependent and that potentially contributesto hypoxic vasodilation. Based on that unexpected discovery, we proposedthat hypoxia-dependent NO production from nitrite in ischemic tissuemight limit ischemia-reperfusion injury. This example provides ademonstration that infusions of sodium nitrite are effective to providecytoprotection during ischemia-reperfusion of the heart and liver.

Although reperfusion of ischemic tissues provides oxygen and metabolicsubstrates necessary for the recovery and survival of reversibly injuredcells, reperfusion itself actually results in the acceleration ofcellular necrosis (Braunwald et al., J. Clin. Invest. 76:1713-1719,1985). Ischemia-reperfusion is characterized by the formation of oxygenradicals upon reintroduction of molecular oxygen to ischemic tissuesresulting in widespread lipid and protein oxidative modifications ofcellular proteins, mitochondrial injury, and tissue apoptosis andnecrosis (McCord et al., Adv Myocardiol 5:183-189, 1985). In addition,following reperfusion of ischemic tissues blood flow may not returnuniformly to all portions of the ischemic tissues, a phenomenon that hasbeen termed the “no-reflow” phenomenon (Kloner et al., J Clin Invest54:1496-1508, 1974). Reductions in blood flow following reperfusion arethought to contribute to cellular injury and necrosis (Kloner et al., JClin Invest 54:1496-1508, 1974). The sudden re-introduction of bloodinto ischemic tissue also results in a dramatic increase in calciumdelivery to the previously ischemic tissue (i.e., “calcium paradox”)resulting in massive tissue disruption, enzyme release, reductions inhigh energy phosphate stores, mitochondrial injury, and necrosis(Nayler, Amer. J. Path. 102:262, 1981; Shen et al., Amer. J. Path67:417-440, 1972). Recent studies have also indicated that theischemia-reperfusion injury is also characterized by an inappropriateinflammatory response in the microcirculation resulting inleukocyte-endothelial cell interactions that are mediated by theupregulation of both leukocyte and endothelial cell adhesion molecules(Lefer et al., Cardiovasc Res 32:743-751, 1996; Entman et al., Faseb J5:2529-2537, 1991). Intensive research efforts have been focused onameliorating various pathophysiological components ofischemia-reperfusion injury to limit the extent of tissue injury andnecrosis.

NO, NO donors, and NO synthase activation or transgenic over-expressionhave been shown to exert protective effects on this process in a numberof models (Lefer et al., New Horiz 3:105-112, 1995; Lefer et al.,Circulation 88:2337-2350, 1993; Nakanishi et al., Am J Physiol263:H1650-1658, 1992; Jones et al., Am J Physiol Heart Circ Physiol286:H276-282, 2004; Jones et al., Proc Natl Acad Sci USA 100:4891-4896.2003; Kanno et al., Circulation 101:2742-2748, 2000), but in othermodels appears harmful (Flogel et al., J Mol Cell Cardiol 31:827-836.1999; Menezes et al., Am J Physiol 277:G144-151, 1999; Woolfson et al.,Circulation 91:1545-1551, 1995; Schulz, R. et al., Cardiovasc Res30:432-439, 1995). Evaluation of these studies suggests a criticaleffect of dose and duration of NO exposure, resulting in a narrowtherapeutic safety window for NO in ischemia-reperfusion pathophysiology(Bolli, J. Mol. Cell. Cardio. 33:1897-1918, 2001; Wink et al., Am JPhysiol Heart Circ Physiol 285:H2264-2276, 2003). An additionallimitation is that NO formation from NO synthase requires oxygen assubstrate, a molecule whose availability becomes limited duringischemia.

We therefore considered the use of nitrite in this context for thefollowing reasons:

-   -   (1) It is a naturally occurring substance with no potentially        toxic “leaving group”,    -   (2) it is selectively reduced to NO in tissues with low oxygen        tension and low pH (Bryan et al., Proc Natl Acad Sci USA, 2004;        Cosby et al., Nat Med 9:1498-1505, 2003; Nagababu et al., J Biol        Chem 278:46349-46356, 2003; Tiravanti et al., J Biol Chem        279:11065-11073, 2004; Doyle et al., J Biol Chem        256:12393-12398, 1981; Luchsinger et al., Proc Natl Acad Sci USA        100:461-466, 2003),    -   (3) its activation does not require molecular oxygen (Cosby et        al., Nat Med 9:1498-1505, 2003), and    -   (4) NO is known to maintain heme proteins in a reduced and        liganded state (Herold et al., Free Radic Biol Med 34:531-545,        2003; Herold et al., J Biol Inorg Chem 6:543-555, 2001;        Fernandez et al., Inorg Chem 42:2-4, 2003), limit free iron and        heme mediated oxidative chemistry (Kanner et al., Arch Biochem        Biophys 237:314-321, 1985; Kanner et al., Lipids 20:625-628,        1985; Kanner et al., Lipids 27:46-49, 1992), transiently inhibit        cytochome c oxidase and mitochondrial respiration (Torres et        al., FEBS Lett 475:263-266, 2000; Brown et al., FEBS Lett        356:295-298, 1994; Cleeter et al., FEBS Lett 345:50-54, 1994;        Rakhit et al., Circulation 103:2617-2623, 2001), and modulate        apoptotic effectors (Mannick et al., Science 284:651-654, 1999),        all mechanisms that might participate in cytotoxicity following        severe ischemia.

We evaluated the effects of nitrite therapy, compared with vehicle andnitrate controls, in well characterized murine models of hepatic andmyocardial ischemia-reperfusion injury. The following descriptionprovides strong evidence for a profound protective effect of nitrite oncellular necrosis and apoptosis, which is believed to be mediated by ahypoxia-dependent bioconversion of nitrite to NO and nitros(yl)atedproteins.

Materials and Methods

Chemicals and Reagents: Sodium nitrite (S-2252) and sodium nitrate(S-8170) were obtained from the Sigma Chemical Co. (St. Louis, Mo.).Sodium nitrite and sodium nitrate were dissolved in phosphate bufferedsaline and the pH was adjusted to 7.4. In all experiments a final volumeof 50 μL of sodium nitrite or sodium nitrate were administered to themice to achieve final concentrations of circulating nitrite of 0.6 to240 μM assuming a total circulating blood volume of 2 mL. Carboxy-PTIO[2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxidepotassium salt], a direct intravascular NO scavenger, was utilized toinhibit NO dependent effects following hepatic I/R injury. Carboxy-PTIO(Alexis Biochemicals) was dissolved in phosphate buffered saline andadministered intravenously at a dose of 1 mg/Kg in a volume of 50 μL at30 minutes prior to hepatic ischemia. Zinc(II) DeuteroporphyrinIX-2,4-bisethyleneglycol (ZnDBG) (Alexis Biochemicals), a hemeoxygenase-1 inhibitor was injected i.p. at a dose of 10 mg/Kg in avolume of 50 μL at 30 minutes prior to the induction of hepaticischemia.

Animals: All of the mice utilized in the present studies were C57BL6/Jat 8-10 weeks of age obtained from the Jackson Laboratories (Bar Harbor,Me.). In additional experiments of hepatic UR injury we utilized micecompletely deficient (−/−) in endothelial nitric oxide synthase (eNOS).eNOS−/−mice were originally generously donated from Dr. Paul Huang(Mass. General Hospital) and generated in our breeding colony atLSU-Health Sciences Center. eNOS−/−mice were utilized at 8-10 weeks ofage.

Hepatic Ischemia-Reperfusion (UR) Protocol: The hepatic UR protocol isdepicted in FIG. 6A and has been described previously (Hines et al.,Biochem Biophys Res Commun 284:972-976, 2001; Hines et al., Am J PhysiolGastrointest Liver Physiol 284:G536-545, 2001). Mice were anesthetizedwith the combination of ketamine (100 mg/kg) and zylazine (8 mg/kg) anda midline laparotomy was performed to expose the liver. Mice were theninjected with heparin (100 μg/kg, i.p.) to prevent blood clotting. Theleft lateral and median lobes of the liver were rendered ischemic bycompletely clamping the hepatic artery and the portal vein usingmicroaneurysm clamps. This experimental model results in a segmental(70%) hepatic ischemia. This method of partial ischemia preventsmesenteric venous congestion by allowing portal decompression throughoutthe right and caudate lobes of the liver. The liver was thenrepositioned in the peritoneal cavity in its original location for 45minutes. The liver was kept moist using gauze soaked in 0.9% normalsaline. In addition, body temperature was maintained at 37° C. using aheat lamp and monitoring body temperature with a rectal temperatureprobe. Sham surgeries were identical except that hepatic blood flow wasnot reduced with a microaneurysm clamp. The duration of hepatic ischemiawas 45 minutes in all experiments, following which the microaneurysmclamps were removed. The duration of hepatic reperfusion was 5 hours inthe studies of serum liver transaminase levels (i.e., AST or ALT) and 24hours for the studies of liver histopathology (such as hepatocellularinfarction).

Liver Enzyme Determinations: Serum samples were analyzed for aspartateaminotransferase (AST) and alanine aminotransferase (ALT) using aspectrophotometric method (Sigma Chemical Co., St. Louis, Mo.) (Haradaet al., Proc Natl Acad Sci USA 100:739-744, 2003). These enzymes areliver specific and are released from the liver during injury (Hines etal., Biochem Biophys Res Commun 284:972-976, 2001; Hines et al., Am JPhysiol Gastrointest Liver Physiol 284:G536-545, 2001).

Liver Histopathology Studies: Histopathology of liver tissue wasperformed as previously reported (Hines et al., Biochem Biophys ResCommit 284:972-976, 2001). Liver tissue was fixed in 10% bufferedformalin for 24 hours, embedded in paraffin, and 10 μM sections stainedwith hematoxylin and eosin. Histopathology scoring was performed in adouble blinded manner on random high power fields using the followingcriteria:

-   -   0—no hepatocellular damage,    -   1—mild injury characterized by cytoplasmic vacuolization and        focal nuclear pyknosis,    -   2—moderate injury with dilated sinusoids, cytosolic        vacuolization, and blurring of intercellular borders,    -   3—moderate to severe injury with coagulative necrosis, abundant        sinusoidal dilation, RBC extravasation into hepatic chords, and        hypereosinophilia and margination of neutrophils,    -   4—severe necrosis with loss of hepatic architecture,        disintegration of hepatic chords, hemorrhage, and neutrophil        infiltration.

Hepatocellular apoptosis was determined using the TUNEL staining kitfrom Roche according to the manufacturer's recommendations. Briefly,liver tissue from various treatments was fixed in buffered formalin and10 μm sections were prepared. Sections were permeabilized on ice for 2minutes and incubated in 50 μL TUNEL solution for 30 minutes at 37° C.Sections were then treated with 50 μL substrate solution for 10 min. andmounted under glass coverslips. The number of apoptotic nuclei wasdetermined from 5 random 40× fields per specimen. A total of sixspecimens per treatment group (16 slides per group) were analyzed andcompared using one-way analysis of variance with Bonferroni'spost-testing.

Myocardial Ischemia-Reperfusion (UR) Protocol: Surgical ligation of theleft main coronary artery (LCA) was performed similar to methodsdescribed previously (Jones et al., Am J Physiol Heart Circ Physiol286:H276-282, 2004). Briefly, mice were anesthetized withintraperitoneal injections of ketamine (50 mg/kg) and pentobarbitalsodium (50 mg/kg). The animals were then attached to a surgical boardwith their ventral side up. The mice were orally intubated with PE-90polyethylene tubing connected to PE-240 tubing and then connected to aModel 683 rodent ventilator (Harvard Apparatus, Natick, Mass.). Thetidal volume was set at 2.2 milliliters and the respiratory rate was setat 122 breaths per minute. The mice were supplemented with 100% oxygenvia the ventilator side port. A median sternotomy was performed using anelectric cautery and the proximal left main coronary artery wasvisualized and completely ligated with 7-0 silk suture mounted on atapered needle (BV-1 ethicon). In the initial experiments of myocardialinfarct size coronary occlusion was maintained for 30-minutes followedby removal of suture and reperfusion for 24 hours. In additionalexperiments of cardiac function, the proximal LCA was completelyoccluded for 45 minutes followed by suture removal and reperfusion for48 hours. In these experiments, two-dimensional echocardiography wasperformed at baseline and again at 48 hours of reperfusion.

Myocardial Infarct Size Determination: At 24 hours of reperfusion, themice were anesthetized as described previously, intubated, and connectedto a rodent ventilator. A catheter (PE-10 tubing) was placed in thecommon carotid artery to allow for Evans Blue dye injection. A mediansternotomy was performed and the left main coronary artery wasre-ligated in the same location as before Evans Blue dye (1.2 mL of a2.0% solution, Sigma Chemical Co.) was injected into the carotid arterycatheter into the heart to delineate the ischemic zone from thenonischemic zone. The heart was rapidly excised and serially sectionedalong the long axis in five, 1 mm thick sections that were thenincubated in 1.0% 2,3,5-triphenyltetrazolium chloride (Sigma ChemicalCo.) for 5 minutes at 37° C. to demarcate the viable and nonviablemyocardium within the risk zone. Each of the five, 1 mm thick myocardialslices were weighed and the areas of infarction, risk, and nonischemicleft ventricle were assessed by a blinded observer usingcomputer-assisted planimetry (NIH Image 1.57). All of the procedures forthe left ventricular area-at-risk and infarct size determination havebeen previously described (Jones et al., Am J Physiol Heart Circ Physiol286:H276-282, 2004).

Echocardiographic Assessment of Left Ventricular Function: Transthoracicechocardiography of the left ventricle using a 15 MHz linear arraytransducer (15L8) interfaced with a Sequoia C256 (Acuson) was performedin additional groups of mice (n=9 vehicle and n=10 nitrite) subjected to45 minutes of myocardial ischemia and 48 hours of reperfusion.Two-dimensional echocardiography was performed at baseline and at 48hours of reperfusion as described previously (Jones et al., Am J PhysiolHeart Circ Physiol 286:H276-282, 2004; Jones et al., Proc Natl Acad SciUSA 100:4891-4896. 2003). Ventricular parameters were measured usingleading-edge technique. M-mode (sweep speed=200 mm/sec) echocardiogramswere captured from parasternal, short and long-axis 2D views of the leftventricle (LV) at the mid-papillary level. LV percent fractionalshortening (FS) was calculated according to the following equation:LV%FS=((LVEDD-LVESD)/LVEDD)×100. All data were calculated from 10cardiac cycles per experiment.

HO-1 Western Blot Analysis of homogenized liver tissue samples (50 μgtotal protein) was performed using mouse anti-HO-1 mAb (Stressgen,Victoria, BC) at a 1:3,000 dilution and goat anti-mouse secondary Ab(Amersham Biosciences, Piscataway, N.J.) at a 1:3,000 dilution.

Blood and Tissue Nitrite Determination: For blood nitrite measurements,160 μL of whole blood was mixed with 40 μL of a nitrite stabilizingsolution containing 80 mM ferricyanide, 20 mM N-ethylmaleimide (NEM),200 μL diethylenetriaminepentaacetic acid (DTPA), and 0.2% NP-40(concentrations provided are after mixing with whole blood). The nitritein whole blood was then measured using tri-idodide-based reductivechemiluminescence as previously described and validated (Gladwin et al.,J Biol Chem 21:21, 2002; Yang et al., Free Radic Res 37:1-10, 2003).

Liver tissue was homogenized using an amended protocol published byBryan and colleagues (Bryan et al., Proc Natl Acad Sci USA, 2004).Harvested liver tissue was blotted dry on filter paper, weighed, andhomogenized immediately in ice-cold NEM (10 mmol/L)/DTPA (2 mmol/L)containing buffer (3:1 dilution—w/v). The buffer/tissue mix was thenhomogenized with a Wheaton glass-glass homogenizer. Tissue homogenateswere kept on ice and analyzed within 5 minutes. The homogenate wassubsequently either injected directly into triiodine to measure the sumof nitrite, mercury stable (Rx-NO) and mercury-labile (RS-NO)NO-adducts. To determine the levels of specific NO-adducts (Rx-NO andRS-NO), the sample was reacted with and without 5 mM mercuric chloride(RS-NO becomes nitrite in presence of mercuric chloride and Rx-NO isstable) and both treated with acid sulfanilamide (0.5%) to eliminatenitrite.

Statistical Analyses: Data were analyzed by two-way analysis of variance(ANOVA) with post hoc Bonferroni analysis using StatView softwareversion 5.0 (SAS Institute, Carey, North Carolina). Data are reported asmeans±standard error of the mean (SEM) with differences accepted assignificant when p<0.05.

Results

Intraperitoneal nitrite limits hepatic ischemia-reperfusion (I/R)injury: Intraperitoneal delivery of 1.2-480 nmoles of sodium nitrite(0.6 μM to 240 μM estimated final concentration in a 2 mL total bloodvolume of the mouse) during hepatic ischemia dose-dependently limitedserum elevations of liver transaminases, aspartate amino transferase(AST) and alanine amino transferase (ALT) (FIGS. 6B and 6C), with a peakeffect occurring at a calculated systemic concentration of 24 μM (48nmoles added nitrite). In sharp contrast, treatment with saline orsodium nitrate (48 nmoles) did not exert any protective effects in thesetting of hepatic I/R injury. Additional studies were performed toevaluate the effects of nitrite treatment on hepatocellular injury inmice following in vivo hepatic ischemia (45 minutes) and more prolongedreperfusion (24 hours; FIGS. 6D, 6E, and 6F). The administration ofnitrite at a final blood concentration of 24 μM (48 nmoles)significantly reduced hepatocellular injury at 24 hours of reperfusioncompared with saline and nitrate treated animals. In addition, nitritetherapy also significantly (p<0.001) attenuated the extent ofhepatocellular apoptosis following 45 minutes of hepatic ischemia and 24hours of reperfusion (FIG. 6F). The extent of hepatic cell apoptosis innitrite treated animals subjected to I/R was similar to that observed insham operated control animals (p=NS).

Intraventricular Nitrite Limits Myocardial Ischemia-Reperfusion Injury:To determine whether the potent cytoprotective effects of nitrite onliver ischemia-reperfusion injury were generalizable to other organsystems, studies were next performed to evaluate the potentialcardioprotective effects of acute nitrite therapy in the setting ofcoronary artery occlusion and reperfusion. The experimental protocol forthe myocardial I/R studies is depicted in FIG. 7A. Administration ofnitrite (48 nmoles) into the left ventricular cavity at 5 minutes priorto reperfusion significantly (p<0.001) limited myocardial infarct size(FIGS. 7B and 7C) compared to 48 nmoles nitrate treatment. Despitesimilar myocardial areas-at-risk (p=NS between groups), myocardialinfarct size per area-at-risk and per left ventricle were both reducedby 67% with nitrite therapy compared to nitrate.

In additional studies, mice were subjected to 45 minutes of myocardialischemia and 48 hours of reperfusion to evaluate the effects of nitritetreatment on left ventricular performance (FIGS. 7D and 7E). In thesestudies, both myocardial ejection fraction (FIG. 7D) and myocardialfractional shortening (FIG. 7E) were measured using two-dimensionalechocardiography at baseline and following myocardial infarction andreperfusion. Myocardial ejection fraction was similar between thevehicle and nitrite treated study groups at baseline. Followingmyocardial infarction and reperfusion, ejection fraction wassignificantly (p<0.001 vs. baseline value) lower in the saline vehiclegroup, yet remained essentially unchanged in the nitrite treated animals(p=NS vs. baseline). Additionally, ejection fraction was significantly(p<0.02) greater in the nitrite group compared to the vehicle group.Similar observations were made for fractional shortening with nosignificant group differences at baseline. However, following myocardialinfarction and reperfusion, left ventricular fractional shortening wassignificantly (p<0.001 vs. baseline) depressed in the vehicle group, butnot in the nitrite group (p=NS vs. baseline) and was significantly(p<0.02) greater in the nitrite group compared to the vehicle group.

Nitrite-Mediated Cytoprotection is Associated with an Acute IschemicReduction of Nitrite to NO and S- and N-nitrosated Proteins within theLiver: Consistent with previously described reduction of nitrite to NOand S-nitrosothiols in a reaction with deoxyhemoglobin and deoxygenatedheme proteins (Bryan et al., Proc Natl Acad Sci USA, 2004; Cosby et al.,Nat Med 9:1498-1505, 2003; Nagababu et al., J Biol Chem 278:46349-46356,2003; Doyle et al., J Biol Chem 256:12393-12398, 1981), one minute afterreperfusion the levels of nitrite in the livers of saline (control)treated mice subjected to ischemia decreased from 1.75 μM toundetectable (p<0.001 vs. sham group) and levels of mercury stable NOmodified proteins (likely N-nitrosamines and iron-nitrosyl proteins;RxNO) increased to approximately 750 nM (FIG. 8A; p<0.001).Interestingly, with nitrite treatment there was a significant (p<0.01vs. saline treated controls) increase in post-reperfusion liver levelsof nitrite (FIG. 8B), S-nitrosothiols (FIG. 8C) and N-nitrosamines (FIG.8D) in the nitrite treated mice. These data are consistent with thethesis that nitrite is bioactivated during hypoxic stress and consistentwith recent studies of Bryan and colleagues demonstrating an acuteconversion of tissue nitrite to RSNO and RxNO after a systemic anoxicinsult (Proc Natl Acad Sci USA, 2004). The low levels of nitrite thatare cytoprotective (1.2 nmoles at lowest dose—FIGS. 6B and 6C) and thereductive decomposition of “native” liver nitrite in the saline treatedcontrol animals (FIG. 8A) suggest that this may be a natural mechanismfor hypoxic NO production and cytoprotection. Consistent with thenear-physiological amounts of nitrite given, blood nitrite levels werenot significantly elevated (594±83 nM to 727±40 nM; n=3; p=0.16) in micetreated with 48 nmoles of nitrite, the most effective dose.

Cytoprotective effects of Nitrite are NO dependent, NO synthaseIndependent and Heme Oxygenase Independent: Further supporting amechanism involving the hypoxic reduction of nitrite to NO, the NOinhibitor PTIO completely inhibited protective effects of nitrite infull factorial design experiments (FIG. 9A). In contrast, significantnitrite cytoprotection was observed in endothelial NO synthase (eNOS)deficient mice (FIG. 9B; p<0.001), suggesting that NO production fromnitrite during ischemia-reperfusion is eNOS independent. While hemeoxygenase 1 protein expression is significantly induced followingischemia-reperfusion in this model, and appears to confer protection(FIGS. 9C and 9D), in mice pre-treated with ZnDPBG (a specific andpotent heme oxygenase 1 inhibitor) nitrite significantly limited tissueinjury suggesting a heme oxygenase-independent effect (FIG. 9C; p<0.05).

Discussion

In this example, nitrite treatment significantly increased the levels ofliver nitrite and nitros(yl)ated species (RSNO and RXNO), compared withsaline and nitrate treated controls, and conferred a dramaticdose-dependent cytoprotective effect, limiting necrosis, apoptosis, andpreserving organ function. Remarkably, the levels of nitrite added werenear-physiological, with a protective effect observed at even 1.2 nmolesadded nitrite (a calculated blood level of 600 nM), suggesting that thismay represent an endogenous protective mechanism that buffers severemetabolic or pathophysiological stress.

Recent data suggest that nitrite concentrations vary between blood anddifferent organs and are typically in the high nanomolar to lowmicromolar range. However, until recently the high concentrationsrequired to vasodilate aortic ring preparations led to its dismissal asan important biologically active molecule. Indeed, Furchgott et al. (J.Pharmaco. Exper. Thera. 108:129-143, 1953) demonstrated in 1953 that 100μM nitrite stimulated vasodilation of aortic ring preparations, aprocess later shown to be mediated by activation of soluble guanylatecyclase (Kimura et al., J Biol Chem 250:8016-8022, 1975; Mittal et al.,J Biol Chem 253:1266-1271, 1978; Ignarro et al., Biochim Biophys Acta631:221-231, 1980; Ignarro et al., J Pharmacol Exp Ther 218:739-749,1981). From a physiological standpoint, the in vivo conversion ofnitrite to NO was thought to be limited to the stomach and severelyischemic heart, where acidic reduction or disproportionation at very lowpH produces gastric mucosal vasodilation (Gladwin et al., J Clin Invest113:19-21, 2004; Bjorne et al., J Clin Invest 113:106-114, 2004) andapparent cardiac tissue injury and heme iron-nitrosylation (at highnitrite concentrations in ischemic ex vivo heart preparations; Tiravantiet al., J Biol Chem 279:11065-11073, 2004), respectively. While xanthineoxidoreductase dependent nitrite reduction can occur at very low oxygentensions, NO production from this system is only detectable in thepresence of high concentrations of superoxide dismutase (Li et al., JBiol Chem 279:16939-16946, 2004; Li et al., Biochemistry 42:1150-1159,2001).

As described in FIG. 6 and Cosby et al. (Nat Med 9:1498-1505, 2003),infusions of sodium nitrite into the human circulation producedsignificant vasodilation at both pharmacological and near-physiologicalconcentrations. The bioactivation of nitrite appeared to be mediated bya nitrite reductase activity of deoxygenated hemoglobin, ultimatelyforming NO and iron-nitrosylated hemoglobin, and to a lesser extentS-nitrosated protein species. Based on these data, a role forcirculating nitrite in mediating hypoxic vasodilation was proposed, withthe oxygen sensor in this case being hemoglobin (Cosby et al., Nat Med9:1498-1505, 2003). It is now proposed that a similar nitrite reductaseactivity of deoxyhemoglobin, deoxymyoglobin and/or other deoxygenatedheme proteins, accounts for the formation of nitros(yl)ated proteins andapparent NO-dependent cytoprotection observed during liver and cardiacischemia in the present example.

Though the precise mechanism of how nitrite confers tissue protection isunclear, a critical role for NO is implicated from data shown in FIGS. 3and 9A. Previous studies of NO and ischemia-reperfusion have yieldedconflicting reports regarding the effects of NO on the severity of I/Rinjury, with some studies suggesting that NO actually contributed toreperfusion injury (Woolfson et al., Circulation 91:1545-1551, 1995;Wink et al., Am J Physiol Heart Circ Physiol 285:H2264-2276, 2003). Ourlaboratory has previously demonstrated that NO donors as well as the NOprecursor, L-arginine, protect against myocardial I/R injury (Lefer etal., New Horiz 3:105-112, 1995; Nakanishi et al., Am J Physiol263:H1650-1658, 1992; Pabla et al., Am J Physiol 269:H1113-1121, 1995).More recently, we demonstrated that the severity of myocardial I/Rinjury is markedly exacerbated in eNOS−/−mice (Jones et al., Am JPhysiol 276:H1567-1573, 1999) whereas mice with eNOS overexpression areprotected against myocardial infarction and subsequent congestive heartfailure (Jones et al., Am J Physiol Heart Circ Physiol 286:H276-282,2004; Jones et al., Proc Natl Acad Sci USA 100:4891-4896. 2003; Jones etal., Am J Physiol 276:H1567-1573, 1999).

Conflicting data on the effects of NO on ischemia-reperfusion injury maybe related to the dose of NO and the conditions during ischemia andreperfusion (Bolli, J. Mol. Cell. Cardio. 33:1897-1918, 2001). It is nowwell appreciated that very high, non-physiological levels of NO (i.e.,high micromolar and millimolar) actually promote cellular necrosis andapoptosis (Dimmeler et al., Nitric Oxide 4:275-281, 1997), while thedemonstrated cytoprotective effects of NO typically involve nanomolar orlow micromolar concentrations of NO (Lefer et al., New Horiz 3:105-112,1995; Lefer et al., Circulation 88:2337-2350, 1993; Bolli, J. Mol. Cell.Cardio. 33:1897-1918, 2001). Additionally, studies investigating NO andNO-releasing agents under in vitro conditions of I/R have consistentlyreported deleterious effects of NO (Bolli, J. Mol. Cell. Cardio.33:1897-1918, 2001), in contrast to in vivo studies of I/R that reportedbeneficial effects of NO therapy (Lefer et al., New Horiz 3:105-112,1995; Lefer et al., Circulation 88:2337-2350, 1993). How NO mediatesprotection is also not clear, with multiple mechanisms being reported,including sGC activation, inhibition of cytochrome C oxidase andinhibition of deleterious mitochondrial calcium uptake (Tones et al.,FEBS Lett 475:263-266, 2000; Brown et al., FEBS Lett 356:295-298, 1994;Cleeter et al., FEBS Lett 345:50-54, 1994; Rakhit et al., Circulation103:2617-2623, 2001). While these data suggest that the effects ofnitrite occur secondary to NO formation, the ultimate mechanism ofnitrite-dependent cytoprotection is currently unknown (Luchsinger etal., Proc Natl Acad Sci USA 100:461-466, 2003; Fernandez et al., InorgChem 42:2-4, 2003; Han et al., Proc Natl Acad Sci USA 99:7763-7768,2002; Crawford et al., Blood 101:4408-4415, 2003).

An intriguing possibility is the intermediate formation ofS-nitrosothiols, known to form via reactions of nitrite withdeoxyhemoglobin and possibly tissue heme proteins (Bryan et al., ProcNatl Acad Sci USA., 2004; Cosby et al., Nat Med 9:1498-1505, 2003;Nagababu et al., J Biol Chem 278:46349-46356, 2003). Consistent withhypoxia dependent formation of S-nitrosothiols in red blood cells andtissues from nitrite, hepatic levels of these species were significantlyhigher following reperfusion (one-to-thirty minutes) in livers exposedto ischemia and nitrite. Within the relative reductive environmentintracellularly, S-nitrosothiols formed via nitrite readily will bereduced to NO and activate sGC. Alternatively, S-nitrosation andsubsequent effects on activity of critical proteins important in I/Rinduced injury and apoptotic cell death may lead to protection (Mannicket al., Science 284:651-654, 1999).

In addition, the data reported here reveal a dynamic regulation ofhepatic RxNO's, a pool of mercury stable NO-modified proteins thatinclude N-nitrosamines and iron-nitrosyls (Bryan et al., Proc Natl AcadSci USA, 2004; Gladwin et al., J Biol Chem 21:21, 2002; Rassaf et al.,Free Radic Biol Med 33:1590-1596, 2002), during ischemia-reperfusion. Insaline treated groups, RxNO levels increase at 1 minutes of reperfusionand then decrease after 30 minutes reperfusion, whereas sustainedelevation in RxNO levels are observed in nitrite treated mice,suggesting that maintenance of RxNO's could be important in protectingtissues from I/R injury.

In conclusion, the data presented in this example demonstrate aremarkable function for the relatively simple inorganic anion nitrite asa potent inhibitor of liver and cardiac ischemia-reperfusion injury andinfarction, as shown in a mouse model system. The effects of nitriteappear NO-dependent, with a rapid conversion of nitrite to NO andnitros(yl)ated proteins following reperfusion. Considering the knownsafety of nitrite as a naturally occurring anion and as an FDA approvedtherapeutic for cyanide poisoning, these data evince a novel, safe, andinexpensive therapy for ischemia-reperfusion injury. Such a therapycould be used to prevent or modulate organ dysfunction following, forinstance, coronary and peripheral vasculature reperfusion, high riskabdominal surgery (such as aortic aneurism repair that leads to renalacute tubular necrosis), cardiopulmonary resuscitation, and perhaps mostimportantly, solid organ transplantation.

Example 3 Inhaled Nebulized Nitrite is a Hypoxia-sensitive NO-dependentSelective Pulmonary Vasodilator

This example provides a description of use of inhaled, nebulized nitrite(specifically, sodium nitrite) to treat neonatal pulmonary hypertension.

Based on the results presented above, it is now known that the bloodanion nitrite contributes to hypoxic vasodilation via a heme-based,nitric oxide (NO) generating reaction with deoxyhemoglobin andpotentially other heme proteins. This biochemical reaction can beharnessed for the treatment of neonatal pulmonary hypertension, anNO-deficient state characterized by pulmonary vasoconstriction,right-to-left shunt pathophysiology, ventilation/perfusion inhomogeneityand systemic hypoxemia. As shown in this example, inhaled sodium nitritewas delivered by aerosol to newborn lambs with hypoxic and normoxicpulmonary hypertension. Inhaled nitrite elicited a rapid and sustainedreduction (˜60%) in hypoxia induced pulmonary hypertension, a magnitudeapproaching that of the effects of 20 ppm NO gas inhalation and whichwas associated with the immediate appearance of increasing levels of NOin expiratory gas. Pulmonary vasodilation elicited by aerosolizednitrite was deoxyhemoglobin- and pH-dependent and was associated withincreased blood levels of hemoglobin iron-nitrosylation. Significantly,from a therapeutic standpoint, short term delivery of nitrite, dissolvedin saline, via nebulization produced selective and sustained pulmonaryvasodilation with no appreciable increase in blood methemoglobin levels.These data support the paradigm that nitrite is a vasodilator acting viaconversion to NO, a process coupled to hemoglobin deoxygenation andprotonation, and further evince a novel, simple and inexpensive therapyfor neonatal pulmonary hypertension.

The effect of nebulized sodium nitrite versus saline, or inhaled NO, onboth hypoxia-induced and drug-induced pulmonary hypertension wascompared in newborn lambs. As described in this example, inhaled nitriteforms expired NO gas and circulating iron-nitrosyl-hemoglobin, andselectively vasodilates the pulmonary circulation. This vasoactivity isassociated with the level of hemoglobin desaturation and blood pH in thephysiologic range, supporting the physiological and therapeutic paradigmof hemoglobin as a deoxygenation-linked nitrite reductase.

Methods

Animal protocols were approved by the Institutional Animal ResearchCommittee of Loma Linda University and were in accordance with theNational Institutes of Health guidelines for use of experimentalanimals.

Animal preparation: Following induction of anesthesia with intravenousthiopental sodium (20 mg/Kg), the newborn lambs were orotracheallyintubated and anesthesia maintained with 1% halothane until catheterswere placed surgically. Thereafter halothane was discontinued andanesthesia maintained with morphine (0.1 mg/kg/hr). After paralysis withvecuronium (0.1 mg/kg/hr) the lungs were mechanically ventilated withinitial settings of pressures: 22/6 cm H₂O, frequency: 25 breaths perminute, FiO₂: 0.21, and inspiratory time: 0.6 seconds (Sechrist Model100, Sechrist Industries, Anaheim Calif., USA). Initially and throughoutthe normoxic experiments, ventilator settings of frequency, peakinspiratory pressure, and FiO₂ were adjusted to maintain SaO₂>95%, PaO₂at 90-150 Torr, and PaCO₂ at 35-45 Torr.

A catheter was placed in the right brachial artery to sample pre-ductalblood for gases and chemical analysis. A pediatric thermodilutioncatheter was passed through a femoral vein to the pulmonary artery tomeasure cardiac output, pulmonary artery and pulmonary capillary wedgepressure (5.0 Pediatric Swan-Ganz® thermodilution catheter, BaxterHealthcare Corporation, Irvine, Calif., USA).

Catheters were placed in the femoral artery and vein for monitoringblood pressure, heart rate, and for administration of fluids and drugs.A thermocouple was placed in the femoral vein to monitor core-bodytemperature which was maintained at 39 C by using a warming blanket andheat lamp throughout the experiments.

After completion of the experiments, the lambs were euthanized with aproprietary euthanasia solution (Euthasol, Western Medical Supply,Arcadia, Calif., USA). In selected experiments necropsy was performed toverify the position of catheters (which were correctly positioned in allcases) and to determine that the ductus arteriosus was closed (which wasclosed in all cases).

Hemodynamic measurements: Mean arterial pressure, mean pulmonary arterypressure, and central venous pressure were measured continuously, andpulmonary capillary wedge pressure was measured intermittently by usingcalibrated pressure transducers (COBE Laboratories, Lakewood, Colo.)zeroed at the midthoracic level. Cardiac output was measured at15-minute intervals throughout the studies by thermodilution using aCom-2 thermodilution module (Baxter Medical, Irvine, Calif., USA).Five-ml injections of ice-cold saline were used. Determinations werecarried out in triplicate and results were averaged for each samplingtime point. Pulmonary vascular resistance and systemic vascularresistance were calculated by using standard formulas.

Blood gas and methemoglobin analysis: Arterial and mixed venous pH,PCO₂, and PO₂ were measured in blood samples (0.3 ml) collected atintervals throughout the experiments. Blood gases were measured (ABL3,Radiometer, Copenhagen, Denmark) and oxyhemoglobin saturation andhemoglobin concentration were measured using a hemoximeter (OSM2Hemoximeter, Radiometer, Copenhagen, Denmark). Arterial and mixed venousmethemoglobin concentrations were analyzed by photometry with the OSM2Hemoximeter using the same arterial sample as in the blood gasdeterminations.

Delivery of aerosolized nitrite, saline, or NO gas: Five milliliters ofeither aqueous sodium nitrite (1 mM solution) or saline were placed in ajet nebulizer (Hudson RCI Micro Mist Nebulizer (Hudson Respiratory Care;Temecula, Calif.), driven at a constant flow rate of 8 L/minute in allexperiments. The sodium nitrite solution was nebulized at a rate of 270μmol/minute. Aerosols were delivered to the inspiration loop of theventilator. Using a jet nebulizer, it is generally thought that <10% ofa nebulized drug deposits in the lung (Coates et al., Chest 119,1123-30, 2001). This is the result of the dead volume of the nebulizerand the loss of drug during the expiratory phase. Lung depositiondepends on particle size distribution, which is under the influence ofair flow, filling volume, drug solution, and ambient temperature (Flavinet al., Pediatr Pulmonol 2, 35-9, 1986; Suarez & Hickey, Respir Care 45,652-66, 2000; Clay et al., Thorax 38, 755-9, 1983; Clay et al., Lancet2, 592-4, 1983). This is a simple, inexpensive, and widely availableclinical nebulizer system, though other systems could be used.

NO gas was introduced into the inspiratory limb of the breathingcircuit. The inspired concentration of NO was continuously measured bychemiluminescence (CLD 700 AL, Eco Physics Inc, Ann Arbor, Mich.) in theinspiratory limb of the ventilator loop.

Inhalation of nitrite or saline aerosols during hypoxic-inducedpulmonary vasoconstriction. Seven lambs were studied in order todemonstrate that nebulized nitrite is a selective pulmonary vasodilatorin hypoxic newborn lambs. After anesthesia and instrumentation, thelambs were allowed to recover for 30 to 90 minutes while relevanthemodynamic parameters were monitored. After baseline measurements wereobtained, a 30-minute period of pulmonary hypertension was induced bydecreasing the FiO₂ of the inspired gas to 0.12 for 30 minutes. Tenminutes after initiation of hypoxia, either saline or sodium nitriteaerosols were administered for the remainder of the hypoxic period.After a one-hour recovery period, a second 30-minute period of hypoxiawas induced again with either saline or sodium nitrite aerosolsadministered during the last 20 minutes. Arterial blood samples forblood gases and analytical assays were drawn and cardiac outputmeasurements were performed at regular intervals.

Inhalation of nitrite during U46619-induced pulmonary hypertension innormoxic conditions. Six additional lambs were studied in order toevaluate the effects of nitrite nebulization on normoxic pulmonaryhypertension. Stable normoxic pulmonary hypertension was induced by aninfusion of a stable endoperoxide analog of thromboxane(U46619-9,11-dideoxy-11α-epoxymethano-prostaglandin F_(2α), CaymanChemicals, Ann Arbor, Mich.). The drug was dissolved in saline and wasadministered at a rate of 2 μg/kg/min into the femoral venous catheterfor 30 minutes. Nitrite was nebulized for inhalation during the last 20minutes of the infusion (FIG. 11).

Comparison of inhaled nitrite and NO gas during hypoxic-inducedpulmonary vasoconstriction: efficacy and duration of effect. Thisprotocol was designed to compare the efficacy of nitrite with theclinical standard, 20 ppm inhaled NO gas. This concentration of NO gasis at the upper end of the therapeutic dose given to infants withprimary pulmonary hypertension (Kinsella & Abman, Semin Perinatol 24,387-95, 2000; Kinsella et al., Lancet 340, 819-20, 1992), and has alsobeen shown to be effective in reversing hypoxic vasoconstriction innewborn lambs (Frostell et al., Circulation 83, 2038-47, 1991). A secondpurpose was to determine the duration of effect of a short nitritenebulization versus NO gas inhalation on hemodynamic and physiologicalmeasurements during prolonged hypoxic-induced pulmonaryvasoconstriction. After baseline measurements were performed, the lambswere made hypoxic as described above for 35 minutes. Ten minutes afterinitiation of hypoxia, a 20-minute period of NO gas inhalation wasinitiated (20 ppm), with continuation of hypoxia for 5 minutes aftercessation of NO gas delivery. Lambs were then allowed to recover for onehour. Again, after baseline measurements were made, a second 90-minuteperiod of hypoxia was initiated. Ten minutes after initiation ofhypoxia, sodium nitrite aerosol was administered for 20 minutes, withcontinuation of hypoxia for 60 minutes after cessation of nitriteaerosolization (FIG. 13).

Measurement of exhaled NO. Exhaled NO concentration was measured with achemiluminescence NO analyzer (NOA 280, Sievers Instruments, Inc.,Boulder, Colo.). The chemiluminescence analyzer was calibrated withNO-free air and NO gas (45 parts per million) according to themanufacturer's recommendations. NO was sampled though a Teflon sidearmattached to a sampling port at the proximal end of the endotracheal tubethrough which flow passed to the analyzer at 250 ml/min.

In selected early experiments, nitrite was nebulized through aventilator circuit with no lamb connected while NO was measured with thechemiluminescence NO analyzer. In no experiments did nitritenebulization through the disconnected circuit result in an increase inNO concentration in the ventilated air.

Measurement of plasma nitrite and iron-nitrosyl-hemoglobin. Blood wasdrawn from both the brachial artery and central venous catheter andrapidly processed. Plasma was separated after centrifugation, frozenimmediately on dry ice, and then stored at −70 C until assayed fornitrite using the chemiluminescence methodologies (Sievers model 280NO-analyzer) as previously described (Cosby et al., Nat Med 9, 1498-505,2003; Gladwin et al., J Biol Chem 277, 27818-28, 2002; Yang et al., FreeRadic Res 37, 1-10, 2003). The frozen red blood cell pellet was thawed,reacted in 8 mM NEM, 100 μM DTPA, and 4 mM ferricyanide, incubated for 5minutes, and passed through a Sephadex G25 column (Yang et al., FreeRadic Res 37, 1-10, 2003; Xu et al., Proc Natl Acad Sci USA 100,11303-8, 2003). The hemoglobin fraction from the G25 column wasquantified by the method of Drabkin (J. Biol. Chem. 112, 51-65, 1935)and reacted in 0.1 M HCl/0.5% sulfanilamide to eliminate residualnitrite. The samples were then injected into a solution of tri-iodide(I₃ ⁻) in-line with a chemiluminescent nitric oxide analyzer (Sievers,Model 280 NO analyzer, Boulder, Colo.). NO gas is striped in thetri-iodide solution stoichiometrically from iron-nitrosyl-hemoglobin(Yang et al., Free Radic Res 37, 1-10, 2003).

Electron paramagnetic resonance spectroscopy of whole blood. This wascarried out at 110K using a Bruker 4131 VT temperature controller on anEMX 10/12 EPR spectrometer system set at 9.4 GHz, 10 mW, 5 G modulation,0.08 s time constant, and 84 s scan time over 600 G. Each curverepresents a single 84-second scan. Concentrations ofiron-nitrosyl-hemoglobin were calculated by comparing the peak-to-peakheights to a standard sample.

Data acquisition and analysis. Mean arterial pressure, pulmonary arterypressure, central venous pressure, heart rate, exhaled NO concentration,and core body temperature were measured continuously. Analog signalswere digitized at 100 Hz and stored using an analogue-to-digitalconverter (PowerLab SP, ADlnstruments, Colorado Springs, Colo.) and dataacquisition software (Chart v 5.02 for Macintosh, ADlnstruments,Colorado Springs, Colo.). Following the experiments, arterial bloodpressure, central venous pressure, heart rate, and exhaled NOmeasurements were averaged into 60-second blocks.

Statistical analysis. Serial measurements of physiological variableswere compared by two-way ANOVA with repeated measures with group andtime as the factors. Significance of differences was evaluated with aDunnett's post-test. Significant differences from the baseline periodwere evaluated using one-way-ANOVA with repeated measures withindividual animals and time as the factors. Significance of differenceswas further evaluated with a Newman-Keul's post-test. The calculationswere done using GraphPad Prism (GraphPad Software Inc., San Diego,Calif., USA). Statistical significance was assumed with P<0.05. Data arepresented as mean±SEM.

Results

Pulmonary Vasodilatory Properties of Aerosolized Nitrite DuringHypoxic-induced Pulmonary Vasoconstriction

In order to determine the effect of nebulized nitrite on hypoxicpulmonary hypertension, seven newborn lambs (2-10 days of age) wereinstrumented under general anesthesia and maintained on mechanicalventilators and morphine infusion. Following baseline stabilization, thelambs were subjected to a 30-minute period of hypoxia by lowering FiO₂to 0.12. Nebulized nitrite or saline was administered for the last 20minutes of the hypoxic period. Initiation of hypoxia (arterial HbO₂˜55%)was associated with rapid increases in mean pulmonary artery pressure(from 21±1 to 34±2 mmHg, P<0.01) (FIG. 10A, 10B) and pulmonary vascularresistance (20% (P<0.01)), and decreased systemic vascular resistance(˜20% (P<0.01)). Inhalation of nebulized nitrite but not saline (FIG.10A, 10B) resulted in a selective decrease in pulmonary artery pressureby ˜60% (P<0.01) (FIG. 10A, 10C) and reduced pulmonary artery resistanceby ˜70% (P<0.05) but had no measurable effect on mean arterial bloodpressure (FIG. 10A, 10C) or systemic vascular resistance when comparedto control animals. The decrease in pulmonary artery pressure withnitrite nebulization was associated with a progressive increase inexhaled NO from 3±1 to 15±4 ppb (FIG. 10A, 10C). Cardiac output,arterial oxyhemoglobin saturation, and methemoglobin levels did notchange measurably after nitrite inhalation as compared to values duringthe preceding ten minutes of hypoxia (FIG. 10A). Arterial PO₂ could notchange appreciably in our system as this was experimentally clamped.

Pulmonary Vasodilating Properties of Aerosolized Nitrite During NormoxicDrug-induced Pulmonary Vasoconstriction

In order to contrast the effects of nebulized nitrite on pulmonaryartery pressure in the presence of normal deoxyhemoglobin with those inthe presence of reduced oxygenated hemoglobin, the effects of nebulizednitrite were studied in a separate group of six lambs subjected topulmonary hypertension under normoxic conditions. Stable normoxic (SaO₂˜98%) pulmonary hypertension was induced by infusion of the endoperoxideanalog of thromboxane (U46619). Intravenous infusion of U46619 at a rateof 2 μg/kg/min for 30 minutes was associated with rapid increases inpulmonary artery pressure from 24±1 to 51±4 mmHg (P<0.001) (FIG. 11).Ten minutes after the infusion began, addition of inhalation ofnebulized nitrite resulted in a selective decrease in pulmonary arterypressure by 23±6% (P<0.05 compared to infusion baseline), but had noeffect on mean arterial blood pressure or systemic vascular resistance(FIG. 11). The decrease in pulmonary artery pressure with nitritenebulization was associated with a progressive increase in exhaled NOfrom 4.8±1.2 to 10.1±2.0 ppb (P<0.05 compared to baseline, FIG. 11).FIG. 2 shows a comparison of the effects of nitrite inhalation after 20minutes on hypoxic versus drug-induced normoxic pulmonaryvasoconstriction. The changes in mean pulmonary artery pressure andexhaled NO were significantly larger with nitrite treatment duringhypoxic conditions. Overall the effects of nitrite inhalation onnormoxic (thromboxane-induced) pulmonary hypertension were less thanthose observed with hypoxic pulmonary hypertension (FIGS. 10, 11, 12A),consistent with a model of hypoxemic and possibly acidemic potentiationof nitrite's vasoactivity.

pH and Oxygen Dependence of the Nitrite Reductase Activity ofDeoxyhemoglobin

We hypothesize that the biochemical conversion of nitrite to NO requiresboth deoxyhemoglobin and protonation. Thus, data from both the normoxicand hypoxic experiments were used to study the influence of hemoglobinsaturation and pH on NO production from nitrite. Measurements of exhaledNO gas and NO-modified hemoglobin (iron-nitrosyl-hemoglobin) were usedas both dosimeters of NO production and as a measure of the directbyproducts of the nitrite reductase reaction of nitrite and hemoglobinto produce NO. FIG. 12 shows that iron-nitrosyl-hemoglobin, measured bytri-iodide based reductive chemiluminescence (FIG. 12B) and electronparamagnetic resonance (FIG. 12C), was markedly increased by nitriteinhalation during hypoxia but not with drug-induced normoxic pulmonaryvasoconstriction. As shown in FIG. 12D, change in mean pulmonary arterypressure during hypoxia after inhalation of nebulized sodium nitrite wasrelated to blood pH, with increased vasodilation associated withdecreasing pH (r=0.57 P=0.055).

Comparison with Inhaled NO and Duration of Effect.

We next compared the efficacy of nitrite with the current therapeuticstandard, inhaled NO gas. After initiation of hypoxia, lambs weresubjected to (20 ppm) inhaled NO gas or nebulized nitrite for 20minutes. The data in FIG. 13 show the duration and magnitude of theeffect of NO gas inhalation (FIG. 13A) or nitrite nebulization (FIG.13B, 13C) on hemodynamic and metabolic measurements during hypoxia.Although both treatments resulted in a pronounced reduction in hypoxicpulmonary hypertension, the response to inhaled NO gas was slightly morerapid and pulmonary pressure more nearly approached baseline whencontrasted to the 60-70% correction in pressure elicited by nitrite.Systemically, mean arterial blood pressure and resistance was reduced toa similar extent with both treatments during hypoxia. However, with therelative chemical stability of the nitrite anion compared with NO gas,there was sustained vasodilation for more than 60 minutes (the durationof the hypoxic challenge) after discontinuation of nitrite inhalation,whereas the termination of NO gas delivery abolished the vasodilatingeffect in a matter of seconds (FIG. 13A, 13B). The relatively sustainedeffect of nitrite nebulization might be therapeutically advantageous byallowing for intermittent therapy analogous to the treatment of asthmawith beta-adrenergic agonists by meter dose inhaler. The time course ofnitrite inhalation-induced pulmonary vasodilation and plasma nitritelevels are shown (FIG. 13C, 13D). In this experiment which trackedbiochemical changes for a longer period that in FIG. 10 methemoglobin(MetHb) concentrations increased from 2.1±0.1% during baseline to2.8±0.2% after nitrite nebulization (P<0.05).

Discussion

A principle finding of this example is that a brief period of inhalationof nebulized sodium nitrite solution produces rapid and selectivepulmonary vasodilation during hypoxic-induced pulmonary hypertension innewborn lambs. The significant reduction in pulmonary artery pressurefollowing nitrite nebulization was sustained when hypoxia was continuedfor more than an hour after termination of nitrite nebulization. In noneof the experiments did nitrite inhalation produce systemic hypotension,and methemoglobin elevation was minimal. From a mechanistic standpoint,nitrite administration was associated with NO production, measured byexhaled NO gas and NO-modified hemoglobin, with responses in proportionto levels of hemoglobin-oxygen desaturation and decreases in blood pH.These data support the paradigm that nitrite is an NO-dependentvasodilator whose bioactivation is coupled to hemoglobin deoxygenationand protonation.

Inhaled NO gas is the current standard for the treatment of pulmonaryhypertension. FIG. 13 provides a comparison of the effects of NO gas at20 ppm with those of aerosolized nitrite. In about 5 minutes the NO gaseffectively ablated about 80% of hypoxic-induced pulmonary hypertension,an effect that was short lived but which could be reproduced when it wasgiven again 20 minutes later. Aerosolized sodium nitrite removed about60% of hypoxic-induced pulmonary hypertension. This response wasconsistently observed in each of the lambs studied and it persistedthroughout the one-hour period of hypoxia that was maintained after thenitrite aerosol was discontinued. The changes in pulmonary blood flowwere accompanied by corresponding changes in the calculated resistanceto blood flow through the lungs, indicating that changes were in thepulmonary vasculature rather than secondary to changes in cardiac outputor systemic effects that might have altered perfusion pressures.

We demonstrate herein that aerosolized nitrite is an NO producing agentin the newborn lamb that can be readily administered by nebulization andappears to exhibit a wide therapeutic-to-safety margin, with limitedsystemic hemodynamic changes and methemoglobin production. This presentsan attractive therapeutic option to inhaled NO. Nitrite is an ideal “NOproducing” agent in that it 1) is a naturally occurring compound inblood, alveolar lining fluid, and tissue, and 2) has no parent-compoundleaving group, such as the diazenium diolates, that requires extensivetoxicological study prior to translation to human disease, and 3) it isalready approved for human use in cyanide antidote kits. Theseadvantages are to be counterbalanced against possible problems thatmight occur with more prolonged delivery, including alveolar nitriteaccumulation, systemic vasodilation, and the development ofmethemoglobinemia.

In conclusion, the data presented in this example suggest that inhalednitrite is a potent and selective vasodilator of pulmonary circulationof the newborn lamb and further support the paradigm that nitrite, andparticularly salts of nitrite, such as sodium nitrite, is anNO-dependent vasodilator whose bioactivation is coupled to hemoglobindeoxygenation and protonation. In none of our studies did inhalingnitrite produce systemic hypotension or elevate methemoglobin levels.

Example 4 Use of Nitrite Infusions for the Prevention of Cerebral ArteryVasospasm after Subarachnoid Hemorrhage

This example describes a method for using nitrite infusion to preventcerebral artery vasospasm after intracranial hemorrhage.

Subarachnoid hemorrhage (SAH) due to the rupture of intracranialaneurysms affects 28,000 Americans annually. Almost 70% of patients withaneurysmal SAH develop severe spasm of the cerebral arteries on theseventh day after SAH. Despite aggressive medical therapy, neurologicaldeficits resulting from vasospasm continue to be a major cause ofmorbidity and mortality. Although the etiology of cerebral vasospasm ispoorly understood, there is increasing evidence that erythrocytehemolysis in the cerebrospinal fluid and decreased availability ofnitric oxide (NO), a potent vasodilator, plays a significant role.Reversal of vasospasm by NO or NO prodrugs has been documented inseveral animal models.

Despite half a century of research and clinical trials, delayed cerebralvasospasm (DCV) remains the single cause of permanent neurologicaldeficits or death in at least fifteen percent of patients followingotherwise successful endovascular or surgical treatment for rupturedintracranial aneurysm. Decreased bioavailability of nitric oxide (NO)has been mechanistically associated with the development of DCV. Thiswork was carried out to determine whether infusions of nitrite, anaturally occurring anion that reacts with deoxyhemoglobin to form NOand S-nitrosothiol, might prevent DCV via reactions with perivascularhemoglobin.

Methods

An autologous arterial blood clot was placed around the right middlecerebral artery (R MCA) of 14 anesthetized Cynomolgus monkeys at day 0.Sodium nitrite solution (NaNO₂, 135 mg/daily and 180 mg/daily, whichapproximates 45 mg/kg and 60 mg/kg per day) in 0.9% saline (n=6) orsaline alone (n=8) was infused intravenously for 14 days in awakeanimals via an ambulatory MiniMed Infusion Pump, at 2 μl/minute.Cerebral arteriogram was performed before clot placement and on days 7and 14, for assessment of DCV. Arteriographic vasospasm was defined as a25% or greater reduction in the proximal 14 mm of the R MCA area asmeasured on the AP projection of the cerebral arteriogram (blindedassessment). Mean arterial blood pressure was measured and blood sampleswere collected daily from day 0; the cerebral spinal fluid samples werecollected on day 0, 7, and 14.

Results

In control animals, cerebral spinal fluid nitrite levels decreased from3.1±1.5 μM to 0.4±0.1 μM at 7 days and 0.4±0.4 μM at 14 days (FIG. 14),and all eight animals developed significant vasospasm of the R MCA(FIGS. 15 and 16), complicated by stroke and death in one animal.

Nitrite infusions were associated with increases in plasma cerebrospinalfluid nitrite and blood methemoglobin concentrations without systemichypotension (FIG. 14), and significantly reduced the severity ofvasospasm (FIGS. 15 and 16; no animals developed significant vasospasm;mean reduction in the R MCA area on day 7 after SAH was 8±9% versus45±5%; P<0.001). Pharmacological effects of nitrite infusion wereassociated with bioconversion of cerebrospinal fluid nitrite toS-nitrosothiol, a potent vasodilating NO donor intermediate of nitritebioactivation. There was no clinical or pathological evidence of nitritetoxicity.

Conclusions

Subacute sodium nitrite infusions prevent DCV in a primate model of SAH,and do so without toxicity. These data evince a novel, safe,inexpensive, and rationally designed therapy for DCV, a disease forwhich no current preventative therapy exists.

While in the foregoing specification this invention has been describedin relation to certain preferred embodiments thereof, and many detailshave been set forth for purposes of illustration, it will be apparent tothose skilled in the art that the invention is susceptible to additionalembodiments, and that certain of the details described herein may bevaried considerably without departing from the basic principles of theinvention.

The invention claimed is:
 1. A method for treating pulmonaryhypertension in a subject in need thereof by decreasing pulmonaryarterial blood pressure or increasing vasodilation, or both, the methodcomprising administering by inhalation a therapeutically effectiveamount of pharmaceutically acceptable non-acidified sodium nitrite tothe subject to decrease the pulmonary artery blood pressure, or increasevasodilation, or both, in the subject, thereby treating pulmonaryhypertension.
 2. The method of claim 1, wherein the sodium nitrite isadministered to a circulating concentration of 0.6 to 240 μM.
 3. Themethod of claim 2, wherein the sodium nitrite is administered to acirculating concentration of about 0.9 μM.
 4. The method of claim 2,wherein the sodium nitrite is administered to a circulatingconcentration of less than 30 μM.
 5. The method of claim 2, wherein thesodium nitrite is administered to a circulating concentration of no morethan about 20 μM.
 6. The method of claim 2, wherein the sodium nitriteis administered to a circulating concentration of no more than about 16μM.
 7. The method of claim 1, wherein the non-acidified sodium nitriteis administered at a rate of 270 μmol/minute.
 8. The method of claim 1,wherein the sodium nitrite is nebulized.
 9. The method of claim 1,wherein the sodium nitrite is administered intermittently.
 10. Themethod of claim 1, wherein the administration of sodium nitrite resultsin minimal methemoglobin elevation.
 11. The method of claim 1, whereinthe administration of sodium nitrite does not produce systemichypotension.
 12. The method of claim 1, wherein the pulmonaryhypertension is primary pulmonary hypertension, secondary pulmonaryhypertension, or both.
 13. The method of claim 1, wherein the pulmonaryhypertension is neonatal pulmonary hypertension.
 14. The method of claim1, wherein the decrease in pulmonary arterial blood pressure or theincrease in vasodilation, or both, in the subject is sustained ascompared to inhaled to nitric oxide.
 15. The method of claim 1, whereinthe subject is human.
 16. The method of claim 1, wherein the sodiumnitrite is administered in combination with at least one additionalagent.
 17. A method for treating pulmonary hypertension in a subject inneed thereof, comprising administering to the subject by inhalation atherapeutically effective amount of non-acidified sodium nitrite.
 18. Amethod for treating pulmonary hypertension in a subject in need thereof,the method comprising administering by inhalation a therapeuticallyeffective amount of pharmaceutically acceptable non-acidified sodiumnitrite to the subject, wherein the sodium nitrite is administered to acirculating concentration of 0.6 to 240 μM, thereby treating pulmonaryhypertension.
 19. The method of claim 18, wherein the sodium nitrite isadministered to a circulating concentration of about 0.9 μM.
 20. Themethod of claim 18, wherein the sodium nitrite is administered to acirculating concentration of less than 30 μM.
 21. The method of claim18, wherein the sodium nitrite is administered to a circulatingconcentration of no more than about 20 μM.
 22. The method of claim 18,wherein the sodium nitrite is administered to a circulatingconcentration of no more than about 16 μM.
 23. The method of claim 18,wherein the sodium nitrite is nebulized.
 24. The method of claim 18,wherein the sodium nitrite is administered intermittently.
 25. Themethod of claim 18, wherein the pulmonary hypertension is primarypulmonary hypertension, secondary pulmonary hypertension, or both. 26.The method of claim 18, wherein the sodium nitrite is administered incombination with at least one additional agent.